Markers of and Risk Factors for the Development and

Progression of Diabetic Kidney Disease

Richard J. MacIsaac, PhD, MBBS, FRACP,1,2 Elif I. Ekinci,2,3,4 and
George Jerums, MBBS, MD2,3

Diabetic kidney disease (DKD) occurs in 25%-40% of patients with diabetes. Given the dual problems of a
significant risk of progression from DKD to end-stage renal disease (ESRD) and increased cardiovascular
morbidity and mortality, it is important to identify patients at risk of DKD and ESRD and initiate protective renal
and cardiovascular therapies. The importance of preventive therapy is emphasized further by worldwide
increases in the incidence of diabetes. This review summarizes the evidence regarding the prognostic value
and benefits of targeting established and novel risk markers for DKD development and progression. Family
history of DKD, smoking history, and glycemic, blood pressure, and plasma lipid level control are established
factors for identifying people at greatest risk of DKD development and progression. Absolute albumin excretion
rate (AER) and glomerular filtration rate (GFR) measurements also are important, although AER categorization
generally lacks the necessary specificity and sensitivity, and estimates of declining GFR are compromised by
methodological limitations for GFRs in the normal-to-high range. Emerging risk markers for progressive loss of
kidney function include markers of oxidation and inflammation, profibrotic cytokines, uric acid, advanced
glycation end products, functional and structural markers of vascular dysfunction, kidney structural changes,
and tubular biomarkers. Among these, the most promising are serum uric acid and soluble tumor necrosis
factor receptor (type 1 and type 2) levels, especially in relation to GFR changes. At present, these can only be
considered as risk markers because they only identify an individual at increased risk of progressive DKD and
not necessarily related to the causal pathway promoting kidney damage. Further work is needed to establish
whether modulating these factors improves the prognosis in DKD. Although change in urinary peptidome
levels also is a promising marker, there currently is neither a clinical assay nor adequate studies defining its
prognostic value. Until these or other novel markers become available for clinical use, predictive accuracy
often may be increased with greater attention to established markers.
Am J Kidney Dis. 63(2)(S2):S39-S62. 2014 by the National Kidney Foundation, Inc.

INDEX WORDS: Diabetes mellitus; end-stage renal disease (ESRD); biomarker; risk factor; disease trajectory.

EXECUTIVE SUMMARY Studies examining the development and progres-

Diabetic kidney disease (DKD) traditionally has
been referred to as diabetic nephropathy. However, the
introduction of a new classication system for chronic
kidney disease (CKD), irrespective of cause, and
increasing awareness of the widening spectrum of
CKD presentations in people with diabetes, beyond the
traditional proteinuria-centered model, have promoted
the use of the term DKD. Classifying patients with
diabetic nephropathy should be reserved for those with
persistent clinically detectable proteinuria that occurs
with elevated blood pressure (BP) and decreased
glomerular ltration rate (GFR). However, subclinical
proteinuria, termed microalbuminuria, has been
recognized as a denable early stage in the natural
history of increasing albuminuria in DKD and some-
times is termed incipient diabetic nephropathy.
Individuals with DKD are at signicant risk of
progression to end-stage renal disease (ESRD) and
cardiovascular morbidity and mortality, emphasizing
the importance of early identication, prevention, and
treatment. The challenge of identifying which patients
will develop progressive DKD is intensied by the
lack of an accepted gold standard for the diagnosis
and progression of DKD.
sion of DKD usually have focused on 3 outcome
parameters: changes in albuminuria, changes in
creatinine level or GFR, and the development of hard
renal end points: death due to kidney disease and/or
the development of ESRD. Caution is required in
interpreting outcomes based on the rst 2 parameters.
Changes in albuminuria have been called into ques-
tion as a surrogate of kidney health outcomes, as
discussed next. It also has been suggested that under
From the 1Department of Endocrinology & Diabetes, St Vin-
cents Hospital Melbourne; 2University of Melbourne, Victoria;
3
Endocrine Centre & Department of Medicine, Austin Health; and
4
Menzies School of Health Research, Darwin, Australia.
Received June 12, 2013. Accepted in revised form October 8,
2013.
This article is part of a supplement that was developed with
funding from Novo Nordisk.
Address correspondence to Richard MacIsaac, PhD, MBBS,
FRACP, Department of Endocrinology & Diabetes, St Vincents
Health, Melbourne, and University of Melbourne, 4th Floor,
Daly Wing, 35 Victoria Parade, PO Box 2900, Fitzroy, Victoria
3065, Australia. E-mail: r.macisaac@unimelb.edu.au
 2014 by the National Kidney Foundation, Inc.
0272-6386/$36.00
http://dx.doi.org/10.1053/j.ajkd.2013.10.048

Am J Kidney Dis. 2014;63(2)(suppl 2):S39-S62 S39


certain circumstances, an increase in serum creatinine
level and hence a decrease in estimated GFR (eGFR)
may represent transient changes in kidney perfusion
or function that are not necessarily related to the
causal pathway that is driving the development of
DKD. From a practical point of view, hard renal end
points usually are worth considering only in large
long-term studies involving high-risk patients with
sufcient events occurring to allow meaningful sta-
tistical comparisons to be made.
Apart from albuminuria and GFR, established or
potential risk markers for DKD include hyperglyce-
mia, hypertension, circulating TNF receptors, hyper-
uricemia, tubular injury markers, presence of specic
urinary peptide classiers, and serum cystatin C level.
However, each of these has limitations and we remain
in need of reliable risk biomarkers to determine which
individuals are at risk of developing DKD that will
progress to ESRD.
The cause of DKD includes irreversible (age, sex,
ethnicity, family history, and duration of diabetes)
and modiable (hyperglycemia, hypertension, albu-
minuria, dyslipidemia, and smoking) risk factors.
Despite evidence of genetic susceptibility and fa-
milial aggregation, associations between particular
gene(s) and indexes of DKD have yet to be reliably
identied. Recent studies have linked specic ge-
netic variants with different DKD phenotypes, but
candidate gene studies and genome-wide linkage
studies have failed to identify a major gene respon-
sible for familial clustering in type 1 and type 2
diabetes, and prospective studies are needed to
investigate which epigenetic factors predispose to
DKD. Once the DKD disease process has started,
metabolic, inammatory, and hemodynamic path-
ways promote progression, sharing a nal common
manifestation of excess accumulated connective tis-
sue, renal brosis, and/or scarring.
Albuminuria has long been used to monitor the
onset and progression of DKD, but microalbuminuria,
historically considered a strong predictor of progres-
sion to proteinuria, recently has been observed to
follow a remission/regression trajectory of less certain
prognostic value. Furthermore, microalbuminuria is
neither a universal nor a sensitive or specic marker
for progressive DKD, and recent studies have chal-
lenged the previously accepted chronological rela-
tionship between albuminuria and GFR decline.
In type 1 diabetes, early accelerated loss of GFR
appears to be a useful predictor of ESRD, whereas in
type 2 disease, decline in GFR often predates mac-
roalbuminuria (although a decline predictive of
ESRD typically depends on progression to macro-
albuminuria). Given this relationship between albu-
minuria and GFR, there now is a move toward using
both, particularly an early decline in GFR above a
S40
MacIsaac, Ekinci, and Jerums
threshold of 60 mL/min/1.73 m2, to identify those at
risk of progressing to advanced kidney disease. In
order to maximize the utility of this strategy, methods
for estimating GFR in the high-normal range will
need to be improved.
Metabolic abnormalities associated with DKD in-
clude hyperglycemia, hypertension, and dyslipidemia.
There appears to be a hemoglobin A1c (HbA1c)
threshold for the development of reduced kidney
function of w6.5% (w48 mmol/mol). Although
glycemic targets should be individualized, intensive
glycemic control clearly has been shown to reduce the
incidence of macro- and microalbuminuria, and recent
evidence indicates that very intensive glycemic con-
trol may slow the decline in GFR and possibly the
progression to ESRD.
There also is a recognized role for hypertension in
accelerating DKD progression, and lowering BP can
slow the deterioration in kidney function. In obser-
vational studies, a continuous relationship between
BP level and kidney and cardiovascular events has
emerged in people with diabetes, although recent
intensive BP-lowering trials have failed to dene the
optimal BP targets for slowing the progression of
kidney and cardiovascular disease. Disturbances in
circadian BP variability are associated with a number
of adverse kidney disease outcomes and may predict
the development of microalbuminuria, DKD pro-
gression, and/or mortality risk.
The role of dyslipidemia remains poorly dened,
although higher levels of LDL (low-density lipopro-
tein) cholesterol and triglycerides and lower HDL
(high-density lipoprotein) cholesterol concentrations
appear to be associated with greater risks of albu-
minuria and declining GFR, and improvement in
these parameters may reduce albuminuria. Statins and
fenobrate may have a renal preservation effect.
Several markers of renal structure and function
have been linked to the development and progres-
sion of DKD. The relationship between structural
changes and functional DKD progression is not
clear cut, and biopsy ndings currently are not
used to determine disease progression. However,
markers of renal vascular function may provide
useful prognostic information. For example, increased
intrarenal vascular resistance and indexes of extra-
renal vascular dysfunction, such as abnormal echo-
cardiogram ndings, are associated with reduced
kidney function. Endothelial dysfunction also is
implicated in the progression of DKD, and asymmet-
rical dimethylarginine (ADMA), the major endoge-
nous inhibitor of nitric oxide production, recently has
emerged as a novel and important predictor of DKD
progression.
A number of urinary and serum markers have been
proposed as risk biomarkers for DKD progression.
Am J Kidney Dis. 2014;63(2)(suppl 2):S39-S62
Markers of DKD Development and Progression
Of these, serum uric acid levels and soluble TNF
receptors seem to be the most promising. Many ob-
servational studies have shown a statistically inde-
pendent association between serum uric acid level and
reduced GFR or the development of proteinuria,
although a relationship between elevated uric acid
levels and microalbuminuria is yet to be determined.
Support for a prognostic role for uric acid level will
require evidence that elevations in uric acid levels
precede a decline in GFR in at-risk patients and
clarication of the effects of lowering uric acid levels
in individuals with diabetes without kidney disease.
Current evidence suggests that serum levels of TNF
receptors (types 1 and 2) may independently predict
progression to ESRD, possibly even with greater
accuracy than albuminuria, although the precise
mechanisms remain unknown, as does the effect of
modulating these receptors. Measurement of the uri-
nary peptidome and specically the CKD273 peptide
classier also shows potential as a biomarker in
INTRODUCTION
It is now well recognized that the incidence of
diabetes is increasing worldwide, mainly due to the
dramatic increase in type 2 diabetes. The public health
impact of this epidemic is vast because diabetes now
is the leading cause of end-stage renal disease (ESRD)
in Western countries. Traditionally, diabetic kidney
disease (DKD) has been referred to as diabetic ne-
phropathy, dened as persistent clinically detectable
proteinuria linked to elevated blood pressure (BP) and
decreased glomerular ltration rate (GFR). Diabetic
nephropathy has been reported to occur in 25%-40%
of people with type 1 or type 2 diabetes. People with
DKD are not only at signicant risk of progression to
ESRD, there also is a concomitant increase in car-
diovascular morbidity and mortality. Thus, it is of
critical importance to identify people at greatest risk
and initiate kidney- and cardiovascular-protective
treatments as early as possible.
This concept has been highlighted in recent studies
that have shown a strong and graded relationship
between indexes of kidney dysfunction and inc-
reased risk for cardiovascular events and all-cause
mortality in people with diabetes. Studies involving
2 separate cohorts of individuals with type 1 diabetes
have indicated that the excess mortality seen in
people with type 1 diabetes is accounted for almost
entirely by those with DKD.1,2 An analysis of ran-
domized controlled trials involving participants with
type 2 diabetes also found a 29-fold difference in
annualized mortality rates, with the highest mor-
tality rates reported from trials involving individuals
with DKD.3 Furthermore, a recent report of a large
Am J Kidney Dis. 2014;63(2)(suppl 2):S39-S62
normoalbuminuric patients with diabetes who are at
risk of progressive DKD.
Other potential signiers of DKD risk and/or pro-
gression require further evidence-based support
before becoming reliable disease markers. Candidates
here include urinary protein-bound advanced glyca-
tion end products, certain markers of oxidative stress,
probrotic cytokines such as connective tissue growth
factor (CTGF) and transforming growth factor b
(TGFb), cystatin C, and tubular markers in the fatty-
acid binding protein family. Novel markers under
investigation include broblast growth factor 23
(FGF-23), urinary type IV collagen, and circulating
and urinary microRNAs.
While awaiting further studies of potential risk
biomarkers for DKD, risk prediction should continue
to focus on family history, smoking history, absolute
(as opposed to categorical) albumin excretion rate,
precise GFR measurements, glycemic control, ambu-
latory BP measurements, and plasma lipid levels.
population-based cohort (1,268,029 participants)
with measures of estimated GFR (eGFR) and pro-
teinuria who were followed up for 4 years showed
that participants with diabetes and proteinuric chronic
kidney disease (CKD), with no history of a previous
myocardial infarction, have incident myocardial
infarction rates that are greater than those for par-
ticipants with a previous myocardial infarction.4
Microalbuminuria is an early step during the pro-
gressive increase in albumin excretion rates (AERs)
that typically characterizes DKD. This subclinical
increase in urinary albumin excretion corresponds to
an AER of 20-200 mg/min (30-300 mg/d) or an
albumin-creatinine ratio (ACR) of 2.5-35 mg/mmol in
males and 3.5-35 mg/mmol in females. The appear-
ance of microalbuminuria usually is regarded as
incipient nephropathy, but it also is a risk factor for
both micro- and macrovascular disease in people with
diabetes, as mentioned. Recently, work has suggested
that some individuals with diabetes and decreased
GFR may not have an increased urinary AER.5 It
therefore is timely that the contemporary approach to
staging DKD now is based on both level of urinary
albumin excretion and eGFR.6
Both absolute change and rate of change in albu-
minuria are linked strongly to the development and
progression of reduced kidney function in diabetes.
Despite this, the sensitivity and specicity of albu-
minuria as a marker of progressive DKD recently has
been challenged, with more emphasis being placed on
early changes in GFR.7 The natural history of DKD in
terms of changes in GFR is shown in Fig 1. Although
the state of hyperltration has been shown to predict
S41

Figure 1. Clinical patterns of diabetic kidney disease.
(A) Normo-hypofiltration; (B) normo-hyper-normo-hypofiltration;
(C) normo-hyper-normofiltration; and (D) persistent normofiltra-
tion. Abbreviation: GFR, glomerular filtration rate.
the progression of albuminuria in many, but not all,
studies, the concept that hyperltration predisposes
to the development of CKD stage 3, that is, a re-
duction in GFR to ,60 mL/min/1.73 m2, remains to
be proved.8
Recently, the prognostic signicance of identi-
fying individuals with diabetes who have an early
(starting at ,60 mL/min/1.73 m2) decline in GFR
(.3.5 mL/min/1.73 m2 per year) that is over and
above what would be expected with aging alone has
been highlighted, with this early decline being linked to
the development of ESRD in type 1 diabetes.9 Apart
from this, established risk factors for DKD are hyper-
glycemia, hypertension, duration of diabetes, age, sex,
smoking, and family history. Emerging risk factors for
the development and progression of DKD include
markers of oxidation and inammation, probrotic
cytokines, uric acid level, advanced glycation end
products (AGEs), levels of markers (both functional
and structural) of vascular dysfunction, kidney struc-
tural changes, and tubular biomarker levels.
This review summarizes the prognostic signicance
of established and novel markers that have been
linked to the development and progression of DKD
in epidemiologic, experimental, and clinical studies.
The potential impact of modulating these markers also
is examined. Despite multifactorial strategies that
target existing risk factors, many individuals with
DKD progress to ESRD. Identifying novel factors
associated with DKD progression may result in
better understanding of the development and progres-
sion of this important diabetes-related complication.
Furthermore, it also may result in the development of
new interventions to prevent or alleviate decreased
kidney function in people with diabetes.
INITIATORS AND PROMOTERS OF DKD
There are several potential initiators and promoters
of DKD, both irreversible and reversible (Box 1). The
S42
MacIsaac, Ekinci, and Jerums
main irreversible factors are age, sex, ethnicity, family
history, and duration of diabetes. Genetic factors in-
uence the development of DKD and are discussed in
more detail in the next section. Although risk for the
development of DKD is inuenced by familial and
ethnic factors, sex also plays a role. In type 2 diabetes,
males are more likely to follow an albuminuric
pathway to decreased kidney function, whereas
females are more likely to follow a nonalbuminuric
pathway.10 Hyperglycemia is the key modiable risk
factor that promotes the development of DKD in both
type 1 and type 2 diabetes. Hypertension classically is
viewed as a promoter of DKD in type 1 diabetes, that
is, it usually only develops after the onset of reduced
kidney function. However, in type 2 diabetes, it most
likely is an important initiator and promoter of DKD.
Albuminuria, dyslipidemia, and smoking are other
well-recognized modiable initiators and promoters
of DKD.
The pathophysiology of DKD is characterized
by the activation of metabolic, inammatory, and
hemodynamic pathways (Fig 2). Metabolic pathways
primarily result from chronic hyperglycemia that
leads to increased protein kinase C (PKC) activity,
abnormalities in polyol metabolism, heightened sec-
retion of probrotic cytokines (such as transforming
growth factor b [TGFb]) and connective tissue
growth factor [CTGF]), nonenzymatic glycosylation
that causes glycation of glomerular and tubular pro-
teins, and the generation of AGEs. From this, reactive
oxygen species (ROS) are produced, and they,
together with an inammatory process, play a key role
in the development of DKD.11,12 The hemodynamic
pathways underlying the development of DKD cause
systemic and intraglomerular hypertension. They
arise by the activation of vasoactive systems such as
Box 1. Initiators and Promoters of DKD
Initiators of DKD

Hyperglycemia

Predisposing genesa
Promoters of DKD

Hyperglycemia

Hypertension

Dyslipidemia

Insulin resistance

Smoking

Procoagulant state

Long duration of diabetesa

Anemia

Ethnicity/westernizationa

Sexa

Agea

Albuminuria
Abbreviation: DKD, diabetic kidney disease.
a
Irreversible risk factors.
Am J Kidney Dis. 2014;63(2)(suppl 2):S39-S62
Markers of DKD Development and Progression

Figure 2. A summary of the postu-

lated contributions of oxidative stress,
growth factor, inflammation, AGEs,
and intraglomerular pressure to the
development and progression of DKD.
(a) Afferent glomerular arteriolar vas-
odilatation 6 systemic hypertension;
(b) angiotensin 2mediated efferent
glomerular arteriolar vasoconstriction.
Abbreviations: AGEs, extracellular
(E/C) and intracellular (I/C) advanced
glycation end products; AMP-k, adeno-
sine monophosphateactivated kinase;
DAG, diacylglycerol; DKD, diabetic
kidney disease; EMT, epithelial mesen-
chymal transformation; eNOS, endo-
thelial nitric oxide synthase; GFR,
glomerular filtration rate; IL-6, interleukin
6; MCP-1, monocyte chemoattractant
protein 1; mTOR-1, mammalian target
of rapamycin; NFkB, nuclear factor-kB;
PKC, protein kinase C; RAGE, receptor
for AGEs; ROS, reactive oxygen spe-
cies; S RAGE, soluble RAGE; TGF-b,
transforming growth factor b.

the renin-angiotensin-aldosterone system (RAAS) (HbA1c, 7.3%; n 5 711) or conventional glycemic

and endothelin system. Of note, these metabolic, in-
ammatory, and hemodynamic pathways can inu-
ence each other. For instance, RAAS activation
promotes endothelial dysfunction, inammation, and
TGFb and CTGF activation.13 In a late stage often
brought about by these pathways, excess connective
tissue accumulates, leading to brosis or scarring of
the kidney.
HYPERGLYCEMIA
DKD requires hyperglycemia to develop, and gly-
cemic control is the primary determinant of the onset
of nephropathy. The DCCT (Diabetes Control and
Complications Trial) in type 1 diabetes and the
UKPDS (UK Prospective Diabetes Study) in type 2
diabetes have revealed a strong relationship between
glycemic control and risk of the development of
diabetic microvascular complications, although there
is no easily dened HbA1c threshold.14,15 However, a
recent sophisticated observational analysis from the
ADVANCE (Action in Diabetes and Cardiovascular
Disease: Preterax and Diamicron Modied Release
Controlled Evaluation) Study, involving participants
with type 2 diabetes, suggested that within the studied
range of HbA1c levels (5.5%-10.5%), there was evi-
dence of a glycemic threshold such that at HbA1c
levels , 6.5%, there was no signicant increase in
risks for the development of microvascular events,
which included the development of eye or kidney
complications, including macroalbuminuria, doubling
of serum creatinine level, need for renal replacement
therapy, or death due to kidney disease.16
In DCCT, participants with type 1 diabetes were
randomly assigned to intensive glycemic control
control (HbA1c, 9.1%; n 5 730) and studied for 6.5
years. At the end of the intervention, intensive therapy
was found to be associated with a signicant reduc-
tion in participants who developed microalbuminuria
(10.2% vs 17.7%, respectively; P , 0.01) or macro-
albuminuria (1.4% vs 3.2%, respectively; P , 0.05).
At the end of a further 16-year observational study,
fewer participants who originally were randomly
assigned to intensive glycemic control compared to
conventional glycemic control developed macro-
albuminuria (7.3% vs 3.2%; P , 0.01). However,
there was no difference in the rate of development of
microalbuminuria.17
In the UKPDS, participants with type 2 diabetes
were randomly assigned to intensive glycemic control
(HbA1c, 7.0%; n 5 2,408) or conventional glycemic
control (HbA1c, 7.9%; n 5 994) and were studied for
10 years. Nine years into the intervention, intensive
therapy was associated with a signicant reduction
in participants who developed microalbuminuria
(19% vs 25%, respectively; P , 0.001) or macro-
albuminuria (4.4% vs 6.5%, respectively; P 5 0.026).
However, at the end of a further 10-year observational
study, participants who originally were randomly
assigned to intensive or conventional glycemic con-
trol had similar levels of albuminuria.18,19
Although there is good evidence to suggest that
elevated glucose levels are an important initiator and
promoter of early DKD and interventions to improve
glycemia can slow the progression of early DKD, to
date, there has been only limited evidence to suggest
that improving glycemia slows the rate of GFR
decline and retards progression to ESRD. However,
evidence is just emerging to suggest that improving

Am J Kidney Dis. 2014;63(2)(suppl 2):S39-S62 S43


glycemia ameliorates a decline in GFR and may even
delay the progression to ESRD.
A recent study from the DCCT/EDIC (Epidemi-
ology of Diabetes Interventions and Complications)
Research Group has shown that long-term risk for
the development of GFR , 60 mL/min/1.73 m2 was
signicantly lower for people who initially were
treated to achieve tight glycemic control.17 A sub-
study from ADVANCE has shown that individuals
with type 2 diabetes randomly assigned to tight,
compared with conventional, glycemic control have a
reduced risk of developing ESRD.20 Furthermore,
preliminary results from the Joslin Diabetes Center
suggest that sustained improvement in glucose control
in patients with type 1 diabetes with overt proteinuria
can reduce the long-term risk of ESRD. A 1%
improvement in HbA1c level over a follow-up of
approximately 3.5 years has been reported to be
associated with a hazard ratio (HR) for ESRD of 0.72
(95% CI, 0.61-0.89), which was independent of other
covariates.21
A meta-analysis of intensive glycemic control
studies in type 2 diabetes has shown that compared
to conventional control, intensive glycemic control
reduces the risk for the development of micro-
albuminuria (risk ratio, 0.86; 95% CI, 0.76-0.96) and
macroalbuminuria (risk ratio, 0.74; 95% CI, 0.65-
0.85). Although risk for the development of ESRD
was reduced by 31% (risk ratio, 0.69; 95% CI,
0.45-1.05), this reduction with intensive glucose
lowering was not signicantly different compared with
conventional glucose control. This result possibly was
related to the relatively low incidence of ESRD
(,1.5%) compared with those of microalbuminuria
(23%) and macroalbuminuria (5%).22
Some of the most impressive information sup-
porting the benecial effects of tight glycemic control
on the kidneys of people with diabetes has come from
a study involving individuals with DKD who received
a pancreatic transplant. After 5 years of normogly-
cemia, achieved by pancreatic transplantation, biopsy
specimens of transplant recipients native kidneys
showed no change in kidney structure. However, after
10 years of normoglycemia, remarkable remodeling
of kidney structure with near-complete reversal of
many of the structural parameters associated with
classic diabetic nephropathy was observed.23
The HbA1c threshold for the development of
reduced kidney function remains to be clearly dened,
but possibly is w6.5%. Achieving this threshold
should be balanced against the increasing apprecia-
tion that glycemic targets for the prevention of
diabetes-related complications need to be individual-
ized for each patient. Tight glycemic control clearly
has been shown to reduce the incidence of micro- or
macroalbuminuria. However, it is only in very recent
S44
MacIsaac, Ekinci, and Jerums
times that some evidence has emerged to suggest that
very intensive glycemic control can slow GFR loss
and possibly the progression to ESRD.
Genetic Markers
Genetic and epigenetic factors may inuence the
development and progression of DKD. Although
there is evidence for genetic susceptibility for the
development of DKD, identication of causative
genes has proved to be elusive. Familial aggregation
of DKD is a well-recognized phenomenon. Diabetic
siblings of patients with ESRD due to diabetes are
known to be at 5-fold higher risk of ESRD compared
with those without a family history.24,25 Despite this
recognized association, many of these high-risk in-
dividuals are not being targeted for early and inten-
sive risk-factor modication. One recent study has
shown that diabetic siblings of patients with ESRD
due to diabetes have a high frequency of albuminuria
(46%), suboptimal BP control (65%), suboptimal
glycemic control (HbA1c . 7.0%; 43%), smoking
(26%), and failure to receive RAAS-modifying agents
(42%).26
Although several candidate DKD genes have been
identied, the association between a particular gene
and indexes of diabetic kidney dysfunction have
not been vigorously replicated. Some of the candi-
date genes for DKD are listed in Box 2. Of all
the candidate genes, those related to RAAS regula-
tion have been studied most extensively. A meta-
analysis of 12 studies examined the relationship
between angiotensin-converting enzyme (ACE) inser-
tion/deletion gene polymorphisms and ESRD risk in
patients with DKD. A signicant association was
found between the deletion allele or homozyg-
ous deletion genotype and ESRD susceptibility. Of
note, these associations were found in the overall
Box 2. Candidate Genes Implicated in the Susceptibility for the
Development of DKD

Angiotensin-converting enzyme (ACE)

Angiotensinogen

Angiotensin II receptor (type 1)

Aldose reductase

Apolipoprotein E

Atrial natriuretic peptide

Heparin sulfate

Intercellular adhesion molecule 1 (ICAM)

Matrix metalloproteinase

Methylene metalloproteinase 9 (MM-9)

Na/H exchanger

Nitric oxide synthase

Plasminogen activator inhibitor 1 (PAI-1)

Peroxisome proliferator-activated receptor (PPAR)

Type 4 collagen

3-Adrenergic receptor

Vascular endothelial growth factor (VEGF)

Engulfment and cell motility 1 (ELMO1)
Am J Kidney Dis. 2014;63(2)(suppl 2):S39-S62
Markers of DKD Development and Progression
population with type 2 diabetes, but not type 1 dia-
betes, and the strongest associations were found
among Asians.27
The more contemporary approach to identifying
genes that are linked to the development of DKD is
to perform a genome-wide association study. This
involves identifying associations between single-
nucleotide polymorphisms and the DKD phenotype
using case-control methodology. Recent studies have
linked specic genetic variants to different DKD
phenotypes. For instance, in type 2 diabetes, gene
variants associated with a decrease in albuminuria
may be different from those associated with an in-
crease in GFR in advanced DKD.28 For patients with
type 2 diabetes, reduced GFR has been linked with
variants in the engulfment and cell mobility (ELMO1)
gene.29 In type 1 diabetes, decreased GFR has been
associated with variants in genes controlling matrix
metalloproteinases and with endothelial nitric oxide
(NO). Despite these advances, both candidate gene
studies and genome-wide linkage studies to date have
failed to identify a major gene that explains the strong
familial clustering of DKD in either type 1 or type 2
diabetes.
Epigenetics is a potential mechanism that links
genes and the environment with DKD.30 Potential
epigenetic markers for DKD progression have been
identied by comparing DNA methylation in in-
dividuals with or without DKD. Different levels of
DNA methylation in individuals with DKD have been
linked with a predisposition to ESRD.31 Of possible
relevance to DKD, methylation and demethylation of
the proximal promoter region of the genes, which
regulate ACE expression, have been shown to upre-
gulate or silence ACE expression.32 However, pro-
spective studies are required to show that epigenetic
factors predispose patients to DKD.
EARLY CHANGES IN GFR
Hyperltration
Hyperltration is a common nding in people with
type 1 diabetes and poor glucose control. There is no
universally accepted denition of hyperltration, but
a common approach is to use a GFR threshold that is
2 standard deviations above the mean GFR in patients
with normal glucose tolerance to signify a state of
hyperltration. In most individuals, this condition
responds to an improvement in glucose control.
However, in a proportion of patients, hyperltration
persists for several years. A recent meta-analysis of 12
studies (6 prospective and 6 retrospective) of patients
with type 1 diabetes and hyperltration concluded
that hyperltration predisposes to the development of
micro- or macroalbuminuria (HR, 2.3) compared to a
concurrent (normoalbuminuric) control group.33 In
Am J Kidney Dis. 2014;63(2)(suppl 2):S39-S62
contrast to the meta-analysis mentioned, a single-
center study from the Joslin Diabetes Center found
no evidence that hyperltration estimated by cystatin
C level predisposes to the development of micro-
albuminuria in type 1 diabetes.34 The exact reasons
for these disparate ndings are not known. However,
it is possible that a calendar effect may explain the
negative ndings in the Joslin Diabetes Center study.
That is, improvements in control of glucose levels,
BP, and dyslipidemia over the last 20 years may have
diminished the role of hyperltration as a predictor of
microalbuminuria.
Studies of hyperltration in type 2 diabetes are
more difcult to interpret than those in type 1 diabetes
because of the effects of age and obesity on GFR. A
correction for age-related loss of GFR can be made
by subtracting 1 mL/min/1.73 m2 per year after 40
years of age. In obese individuals with type 2 dia-
betes, there also is a continuing debate about whether
GFR should be indexed for body surface area. In a
study involving Pima Indians with type 2 diabetes
with average age younger than 50 years, early GFR
loss ($3.3% per year) occurred over 4 years in 88
participants and kidney function remained stable in
107 participants. Hyperltration at baseline, dened
as GFR $ 154 mL/min, was present in 53% of par-
ticipants with early GFR loss and a similar proportion
of participants with stable kidney function (48%).
Therefore, this study does not support the concept that
hyperltration predisposes to early GFR decline.35
However, it should be noted that mean body mass
index (BMI) was .30 kg/m2 in both study groups,
and if GFR were expressed per body surface area,
most of these individuals would not be classied as
having hyperltration.
The risk of the development of micro- or macro-
albuminuria in hypertensive individuals with type 2
diabetes, with or without persistent hyperltration,
has been examined over a 4-year follow-up period.
From the 90 individuals with hyperltration at base-
line, 47 still displayed a state of hyperltration after
a further 6 months of observation. Of these in-
dividuals, 11 (23.4%) progressed to the development
of micro- or macroalbuminuria compared with 53 of
the 502 (10.6%) individuals for whom hyperltration
was ameliorated at 6 months or who had normol-
tration at baseline (HR, 2.16; 95% CI, 1.13-4.14).36
Due to the short study duration, it was not possible
to determine whether amelioration of hyperltration
results in slower progression to a GFR threshold of
60 mL/min/1.73 m2.
In summary, to date, there is no evidence that
hyperltration predicts the development of
GFR , 60 mL/min/1.73 m2. Further long-term stu-
dies of GFR gradients therefore are required to
establish whether hyperltration ultimately leads to
S45

decreased kidney function, after adjustment for gly-
cemic control and other confounders. Although the
state of hyperltration has been shown to predict the
progression of albuminuria in many, but not all,
studies, the concept that hyperltration predisposes to
the development of CKD stage 3, that is, GFR decline
to ,60 mL/min/1.73 m2, remains to be proved.8
Early Kidney Function Decline
For type 1 diabetes, early decline in kidney func-
tion is dened as a yearly decrement in GFR . 3.3%;
this corresponds to the 97.5 percentile of the decline
in creatinine clearance in nondiabetic normotensive
whites in the Baltimore Longitudinal Study on Ag-
ing.37 Using this denition, the rst Joslin Kidney
Study followed up trends in kidney function over
4 years in 279 participants with type 1 diabetes with
new-onset microalbuminuria. To compare the annual
percentage of change in GFR in this cohort (mean
baseline GFR, 155 mL/min/1.73 m2) with a control
group of normoalbuminuric patients (baseline GFR,
143 mL/min/m2), the authors calculated the reciprocal
of serum cystatin C concentration. There was early
kidney function decline in 31% of microalbuminuric
and 9% of normoalbuminuric patients (P , 0.001).38
The frequency of early kidney function decline was
related to the trajectory of AERs: a higher frequency
of early GFR loss (67.7%) was seen in participants
for whom AERs doubled during the study compared
with those with stable microalbuminuria (32.2%)
and participants with halving of microalbuminuria
(16.2%). The implication of these ndings is that
early GFR decline and AER trends are separate pro-
cesses at an early stage, but become more closely
coupled at later stages of DKD.
In a long-term analysis of the DCCT/EDIC
cohort performed over 15 years in participants with
type 1 diabetes, a persistent decline in eGFR to
,60 mL/min/1.73 m2 was observed in 89 of 1,439
participants, of whom 21 had normoalbuminuria at
baseline. When expressed per 1,000 person-years,
this translated to 36.1 macro-, 1.3 micro-, and 1.0
normoalbuminuric participants progressing to CKD
stage 3.39 The prognostic signicance of early GFR
loss in type 1 diabetes has been highlighted in a
recent study from the Joslin Diabetes Center involving
proteinuric patients. An early accelerated loss of
GFR, which occurred in approximately two-thirds
of patients, was found to be a better predictor of
ESRD development over a 5-year observational period
than HbA1c, BP, and ACR values at baseline. In
contrast, for patients without an early accelerated
decline in GFR, risk for the development of ESRD was
considered to be negligible.9
The relationship between early renal function loss
and AER also has been studied in type 2 diabetes. In a
S46
MacIsaac, Ekinci, and Jerums
long-term study of 195 Pima Indians with type 2
diabetes, early kidney function decline was dened as
a rate of GFR loss $ 3.3% per year over 4 years. In
this study, GFR was measured directly by urinary
clearance of iothalamate and expressed as milliliters
per minute (no correction for body surface area).
The prevalence of early kidney function decline
during this initial period was 32% in participants
with normal urinary ACRs at baseline, 42% in those
with microalbuminuria, and 74% in those with mac-
roalbuminuria (P , 0.001). Subsequent follow-up
showed that the cumulative incidence of ESRD 10
years after the initial period was 41% in those with
early kidney function decline and 15% in those
without. It was concluded that an early decline in
GFR often happens before macroalbuminuria occurs,
but a decline predictive of ESRD is highly dependent
on the development of macroalbuminuria.35
ALBUMINURIA
Prognostic Signicance of Progression and Remission
of Microalbuminuria
Studies dating back to the early 1980s reported that
microalbuminuria is associated with a 60%-80% risk
of the development of overt proteinuria over 6-14
years in patients with type 1 diabetes. However, more
recent studies have raised questions about the prog-
nostic signicance of microalbuminuria. The issue was
raised rst following a pooled analysis by Caramori
et al40 that demonstrated that the cumulative incidence
of remission of microalbuminuria varies from 0%-43%
in type 1 diabetes, and that rates of progression to
macroalbuminuria are only w30%. Other studies
have detected microalbuminuria remission rates of
21%-64% in patients with type 1 or type 2 diabetes.5
In some, but not all, studies showing substantially
higher rates of microalbuminuria remission, ndings
have been linked to the use of RAAS blockers. This
may be explained by the almost universal treatment
of patients with microalbuminuria with these agents,
the observational design of most of the studies, and
insufcient documentation of persistent microalbu-
minuria at baseline in some studies. Therefore, in the
present treatment environment, microalbuminuria is
linked more frequently to remission/regression rather
than the development of overt proteinuria.
In results from the STENO-2 study, intensive
multifactorial intervention was associated with a
lower rate of progression from microalbuminuria to
overt nephropathy over 8 years in participants with
type 2 diabetes. However, the rate by which isotopi-
cally measured GFR declined was similar whether
participants received conventional or intensive multi-
factorial intervention. A relationship between decline
in AER and slower pace of GFR decline was apparent
Am J Kidney Dis. 2014;63(2)(suppl 2):S39-S62
Markers of DKD Development and Progression
in only a post hoc analysis that looked at pooled
results from groups treated by the conventional or
intensive strategies.41 Similarly, a post hoc analysis of
the IRMA-2 (Irbesartan in Patients With Type 2
Diabetes and Microalbuminuria) trial has shown that
reductions in albuminuria within the microalbuminuric
range are linked to a slower decline in eGFR. However,
this relationship has been demonstrated only when
the albuminuria and GFR relationship was analyzed
after combining irbesartan- and placebo-treated in-
dividuals.42 In another observational study of micro-
albuminuric individuals with type 1 diabetes, an early
decrease in GFR happened in 68%, 32%, and 16% of
participants with AER progression, lack of change, or
regression, respectively, over the 8- to 12-year obser-
vation period.38
As opposed to the observational studies mentioned
in the preceding, a pooled analysis of interventional
studies has demonstrated that the initial decline in
AER with antihypertensive therapy is not a predictor
of the subsequent decrease in GFR during the
microalbuminuric phase for patients with type 1 or
type 2 diabetes. In contrast, AER and GFR responses
in patients with type 1 or type 2 diabetes and late
nephropathy were found to be closely correlated.
These data suggest that the full relationship between
current therapies, microalbuminuria, and GFR still
needs to be elucidated.43 On this basis, the value of
microalbuminuria as a kidney disease end point in
clinical trials has come under question.
Despite this, observational studies have shown a
continuous relationship between increasing albumin-
uria and declining GFR. This relationship becomes
evident for increasing albuminuria even before the
upper limit of normoalbuminuria is reached.44
Furthermore, albuminuria within the microalbumi-
nuric range has been shown to be associated with
increased risk for the development of ESRD
compared with normoalbuminuria in a meta-analysis
of 8 studies with an overall relative risk for ESRD
of 4.8 (95% CI, 3-7.5). In a similar fashion, a meta-
analysis of 5 studies involving individuals with type
2 diabetes found that microalbuminuria was associ-
ated with a relative risk of 3.6 (95% CI, 1.6-8.4)
for the development of ESRD compared with
normoalbuminuria.45
When interpreting these results, it is salient to
consider the limitations of meta-analyses or pooled
analyses that include reliance on the quality of the
individual studies included in the analysis, the fact that
grouped and not individual patient data usually are
analyzed, the heterogeneity of the individual studies,
and the possible inuence of publication bias.46
The best method for measuring albuminuria,
immunoassay or high-performance liquid chroma-
tography (HPLC), has become an issue of debate.47
Am J Kidney Dis. 2014;63(2)(suppl 2):S39-S62
HPLC overestimates AER in the normo- to micro-
albuminuric range. For example, in community
studies such as PREVEND (Prevention of Renal and
Vascular End-Stage Disease) and AusDiab (Austra-
lian Diabetes, Obesity and Lifestyle), similar in-
creases in HPLC-assayable albumin were seen in
participants with normoalbuminuria regardless of
whether they also had diabetes.48,49 In healthy in-
dividuals, the albumin peak on size-selective HPLC
consists of w70% albumin, with the remaining
30% reecting globulins such as transferrin and
a1-glycoprotein. Use of HPLC-assayed albumin as a
marker of progression would require determining
appropriate thresholds based on HPLC-measured
albuminuria that would allow the transition from
normoalbuminuria to micro- and macroalbuminuria to
be recognized in long-term studies.
Dissociation Between Albuminuria and GFR
In the past decade and a half, worsening AER as a
denitive predictor of diabetic CKD has come under
re. First, spontaneous remission of microalbuminuria
has been reported in .50% of patients with diabetes,
as described previously. Second, early GFR decline
now can be detected at the time of incident micro-
albuminuria, substantially before the onset of overt
nephropathy. These studies have been facilitated by
the use of markers such as cystatin C that accurately
identify a decline in GFR even when the resulting
level is still . 60 mL/min/1.73 m2. In a recent study
from the Joslin Diabetes Center, 79 patients with type
1 diabetes, new-onset microalbuminuria, and baseline
eGFR of 104 mL/min/1.73 m2 were followed up over
a 12-year period. In that time, 23 of 79 patients
reached eGFRs , 60 mL/min/1.73 m2 (CKD stage 3).
Of these, 12 had AERs at the macroalbuminuria stage,
but the occurrence of macroalbuminuria was con-
temporaneous with, not before, the development of
advanced CKD. In the other 11 participants, micro-
albuminuria persisted or reverted to normoalbumi-
nuria although they reached CKD stages 3-5.50
The third strike against AER as a prognostic indi-
cator comes from 2 studies that appeared in the early
1990s, which found that some patients with diabetes
develop a low creatinine clearance (CCr) while never
progressing beyond normoalbuminuria. The rst
study, involving kidney biopsies in type 1 diabetes,
found changes of classic diabetic glomerulopathy in a
small group of female patients with reduced CCr
(,90 mL/min/1.73 m2) and normal AER.51 The sec-
ond publication, a study of serial kidney function using
CCr, showed that w30% of diabetic patients (both
type 1 and type 2) have progressive declines in CCr
without going from normo- to microalbuminuria.52
That study involved 40 patients followed up over
8-14 years who had normal AERs and CCrs at baseline.
S47

Three patterns of AER and CCr became evident. In 15
patients, AER increased and CCr was stable; in 13,
AER increased and CCr decreased; and in 12, AER
stayed in the normoalbuminuric range but CCr
declined.52 Dissociation between AER and GFR also
has been reported in type 2 diabetes.53,54
The normoalbuminuria/low-GFR pattern occurs
more frequently in elderly women. Furthermore,
separate sets of risk factors been reported in recent
studies for progression to low GFR versus the devel-
opment of increased AER; this provides additional
evidence that an increase in AER and decrease in GFR
are complementary rather than necessary manifesta-
tions of diabetic CKD. In the UKPDS-74, independent
risk markers for decreased GFR were female sex,
smaller waist circumference, age, increased insulin
sensitivity, and previous sensory neuropathy. How-
ever, male sex, larger waist circumference, plasma
triglyceride level, low-density lipoprotein (LDL)
cholesterol level, HbA1c level, increased white blood
cell count, history of smoking, and previous retinopathy
were independent risk markers for elevated AER.10
HYPERTENSION
In individuals with type 1 diabetes, the cause of
high BP generally is DKD, and usually BP starts to
increase only when the patient progresses from micro-
to macroalbuminuria. However, hypertension is found
in about one-third of patients with type 2 diabetes at
the time of diagnosis. Thus, for type 2 diabetes, hy-
pertension most likely is caused by many factors and
may represent a component of metabolic syndrome.
Despite the differences in timing and cause of hy-
pertension in type 1 and type 2 diabetes, a number of
studies have shown that high BP hastens the devel-
opment of DKD and that reducing BP slows the loss
of kidney function.
Although the evidence from observational studies
is consistent with a continuous relationship bet-
ween BP levels and renal and cardiovascular events
in patients with diabetes, very few interventional
studies have succeeded in attaining BPs below the
130/80mm Hg threshold (or the stricter target
of ,120/75 mm Hg if proteinuria is present). In both
the ACCORD-BP trial and INVEST (Interna-
tional Verapamil-Trandolapril Study), which achieved
BPs , 130/80 mm Hg, the results suggested that
further lowering BP does not substantially reduce
clinical events versus moderate BP control (equiva-
lent to w135/85 mm Hg).55,56 Of note, the American
Diabetes Association has recently relaxed its general
systolic BP goal from ,130 to ,140 mm Hg.57
Data from ambulatory BP monitoring may be of
value in estimating the risk of clinical outcomes,
including kidney events in diabetes. Such data include
estimating true BP levels and detecting the white-
S48
MacIsaac, Ekinci, and Jerums
coat effect; measurement of the diurnal rhythm of
BP, by which patients can be classied into nocturnal
dippers and nondippers (reduction in BP , 10%);
and recording BP random variability and ambulatory
heart rate. An abnormal circadian variability in BP in
individuals with diabetes has been linked to a number
of kidney disease outcomes. Nondipping of night-
time BP values predicts the development of micro-
albuminuria in individuals with type 1 diabetes.58
Individuals with nondipping of night-time BP who
have type 1 or type 2 diabetes also are at higher risk
of mortality compared with dippers, even after ac-
counting for traditional cardiovascular disease risk
factors.59 Recently, higher 24-hour systolic BP and
pulse pressure values and reduced night/day BP
variation have been shown to be independent pre-
dictors of DKD progression, dened by increases in
AER and/or doubling of serum creatinine level or the
development of end-stage renal failure in individuals
with type 2 diabetes.60
On the basis of receiver operating characteristic
curve analysis, one cross-sectional study of 550 par-
ticipants with type 2 diabetes suggested that the
ambulatory BP threshold levels that were associated
with overt proteinuria were 125/75, 110/65, and
120/75 mm Hg for daytime, night-time, and 24-hour
periods, respectively.61
DYSLIPIDEMIA
The role of dyslipidemia in the initiation and pro-
gression of DKD still is poorly dened. Results of a
number of cross-sectional and prospective studies
reveal associations between dyslipoproteinemia, in
particular elevated apoB-100containing lipoprotein
levels, low high-density lipoprotein (HDL) cholesterol
levels, and albuminuria in individuals with diabetes.
In a post hoc analysis from the ADVANCE Study,
low baseline HDL cholesterol levels were associated
with a signicantly higher risk for the development of
new microalbuminuria and macroalbuminuria. An
effect in the same direction was seen for the outcomes
of doubling of creatinine level and death related to
kidney disease, but it was not statistically signicant.
Interestingly, no association was detected between
baseline HDL cholesterol levels and the development
of retinopathy.62 Furthermore, the use of fenobrate
appears to protect against eGFR loss to a greater extent
in individuals with low HDL cholesterol and higher
triglyceride levels, whereas the retinal benets of
fenobrate appear to be independent of lipid levels. In
contrast, in the DCCT/EDIC cohort, HDL cholesterol
concentrations were not associated with albuminuria,
but smaller HDL cholesterol particles were associated
positively with increasing albuminuria.63
Regression of microalbuminuria has been shown to
be associated independently with low levels of LDL
Am J Kidney Dis. 2014;63(2)(suppl 2):S39-S62
Markers of DKD Development and Progression
cholesterol and triglycerides in studies from the
Joslin Diabetes Center and higher LDL cholesterol
and triglyceride levels were associated with inc-
reasing albuminuria in the DCCT/EDIC cohort.63,64
Furthermore, declining GFR (as measured by CCr)
has been linked with increasing LDL cholesterol
plasma levels, smaller LDL cholesterol size, and
elevated triglyceride levels in the DCCT/EDIC
mentioned previously. Studies involving the Finne-
Diane cohort have suggested that higher triglyceride
levels are associated with progressive albuminuria,
whereas total cholesterol was the only lipid parameter
associated with progression to ESRD in an adjusted
Cox regression model that included HbA1c level, sex,
smoking, BP, BMI, duration of diabetes, and eGFR.65
Randomized controlled trials have suggested that
statins may preserve kidney function and that a
greater degree of renal preservation is seen for in-
dividuals treated with higher, compared with lower,
doses of statins who also achieved the greatest re-
ductions in LDL cholesterol and triglyceride levels.66
In CARDS (Atorvastatin Study for the Prevention of
Coronary Artery Disease Endpoints in Non-Insulin
Dependent Diabetes Mellitus), treatment with ator-
vastatin was found not to be associated with pro-
gression or regression of albuminuria, but slowed the
annual change in eGFR (w0.2 mL/min/1.73 m2 per
year; P 5 0.01) compared with placebo.67
STRUCTURAL MARKERS
Kidney Size
Diabetic hypertrophy has been linked to the
development of microalbuminuria and increased risk
of developing ESRD when compared with normal-
sized kidneys.68,69 Kidney hypertrophy most likely
represents a maladaptive response to hyperglycemia
and the release of various cytokines and growth fac-
tors, such as insulin-like growth factor 1 (IGF-1),
TGFb, vascular endothelial growth factor (VEGF),
and possibly through adenosine monophosphate
(AMP)-activated protein kinase, a key player in
regulating kidney metabolism that predisposes to the
development of DKD.70
Kidney Biopsy Findings
The characteristic morphologic changes that are
seen in type 1 DKD are glomerular basement mem-
brane thickening, mesangial expansion, and hyali-
nosis of the afferent and efferent arterioles. Pathologic
changes in glomerular epithelial cells that are asso-
ciated with decreased number of podocytes and/or
podocyte foot-process broadening and effacement
also are seen. The major glomerular feature that pre-
dicts DKD progression is mesangial expansion. It
therefore is the central morphologic feature that forms
Am J Kidney Dis. 2014;63(2)(suppl 2):S39-S62
the basis of the new pathologic classication system of
diabetic nephropathy.71 However, although morpho-
logic changes such as mesangial expansion and
glomerular basement membrane thickening can be
linked to markers of DKD progression such as
increasing albuminuria and declining GFR, dissocia-
tion between structure and function has been described.
When persistent microalbuminuria occurs in type 1
diabetes, kidney changes detected on biopsy often are
advanced.72 There also is a great deal of heterogeneity
in biopsy ndings from kidneys of individuals with
type 2 diabetes and microalbuminuria.73
Furthermore, a recent study of proteinuric patients
with type 2 diabetes and biopsy-proven mesangial
expansion showed that glomerular changes were not
associated with the outcome of initiation of mainte-
nance dialysis therapy or doubling of serum creatinine
level. In contrast, interstitial changes such as brosis,
tubular atrophy, and interstitial inammation were
associated independently with these kidney disease
end points.74
In summary, although some morphologic changes
detected on kidney biopsy are linked to functional
changes, the DKD pathologic ndings that predict
kidney disease outcomes still remain to be clearly
dened. Therefore, the use of biopsy ndings as a
marker of disease progression is still not recognized
as a part of routine clinical practice for the manage-
ment of DKD.
Structural and Functional Indexes of Vascular
Dysfunction
Patients with type 2 diabetes and eGFR , 60 mL/
min/1.73 m2 have higher measures of intrarenal
vascular resistance, as measured by Doppler ultra-
sound techniques, compared with patients with
eGFR $ 60 mL/min/1.73 m2. However, renal vascular
resistance index is elevated to a similar extent in pa-
tients with type 2 diabetes and eGFR , 60 mL/min/
1.73 m2, regardless of albuminuric status (Fig 3).75
Higher, compared to lower, baseline indexes of intra-
renal vascular resistance also are associated with a
greater subsequent decline in GFR and likelihood for
the development of proteinuria in type 2 diabetes.76
Extrarenal vascular dysfunction, as measured by
abnormal echocardiogram ndings and mainly related
to the nding of diastolic dysfunction, has been
associated with reduced kidney function as measured
by CCr in individuals with type 2 diabetes.77
Increased aortic stiffness, as assessed by pulse wave
velocity, also has been linked to the development of
more advanced stages of albuminuria and annual
change in GFR.78 Further evidence of extrarenal
vascular disease being linked to reduced kidney
function in diabetes has been provided by a study
using magnetic resonance imaging to detect silent
S49

Figure 3. Intrarenal arterial resistance index in 325 patients
with type 2 diabetes stratified according to eGFR (ie, ,60
or $60 mL/min/1.73 m2) and albuminuric status (ie, normoalbu-
minuria [AER , 20], microalbuminuria [20-200], or macroalbumi-
nuria [.200]). Open bars: eGFR , 60 mL/min/1.73 m2 (n 5 93);
closed bars: eGFR $ 60 mL/min/1.73 m2 (n 5 232). Repro-
duced with permission of the American Diabetes Association
from MacIsaac et al.75
cerebral infraction, a marker of small-vessel disease.
In that study, multivariate analysis showed that the
composite outcome of a doubling of serum creatinine
level or commencing dialysis therapy was signi-
cantly greater for individuals with evidence of silent
cerebral infraction compared with those without,
during a follow-up of 7.5 years. Interestingly, the
presence of silent cerebral infraction did not increase
the risk for the progression of albuminuria.79
Endothelial dysfunction usually is characterized by
an imbalance between vasodilating and vasocon-
stricting substances, either produced by or acting
on the endothelium, but also related to perturba-
tions of other endothelial functions, including those
related to coagulation, platelet adhesion, and immune
function. A quantiable feature of endothelial dys-
function is the inability of arteries to dilate fully in
response to appropriate stimuli. NO, generated by
the endothelium, is a key substance that regulates
vasodilation, with asymmetrical dimethylarginine
(ADMA) being the major endogenous inhibitor of
NO production.80
One recent study has suggested that ADMA may
be a novel and important predictor of DKD progres-
sion. In that study, 225 participants with type 2 dia-
betes and with the majority having normoalbuminuria
and eGFR . 60 mL/min/1.73 m2 had baseline levels
of ADMA measured and then were followed up for 5
years. Those with higher, compared with lower,
baseline ADMA levels had a greater chance of pro-
gressing to a more advanced stage of albuminuria and
also had a greater rate of decline in eGFR during
follow-up. Furthermore, the relationship between
baseline ADMA levels and increases in albuminuria
S50
MacIsaac, Ekinci, and Jerums
and decreases in GFR was independent of established
risk markers of DKD.80
Biomarkers of endothelial dysfunction that are not
directly related to the vasoconstriction-vasodilation
process also have been implicated in the progression
from microalbuminuria to macroalbuminuria. Using
a combined z score for 4 markers of endothelial
dysfunction (von Willebrand factor, soluble vascular
cell adhesion molecule 1 (VCAM-1), soluble inter-
cellular cell adhesion molecule 1 (ICAM-1), and
soluble E-selectin), it has been shown that a 1standard
deviation increase in z score is associated with an HR
of 3.2 (95% CI, 1.56-6.56) for the development of
macroalbuminuria that is independent of established
risk markers. In addition, endothelial dysfunction z
score was found to enhance risk assessment for the
development of macroalbuminuria when combined
with albuminuria and whether participants were treated
with an RAAS blocker.81
ADVANCED GLYCATION
The accumulation of abnormal indicators of pro-
tein glycation is thought to play a key role in the
initiation and progression of DKD. Immunohisto-
chemical studies in individuals with DKD have
shown that AGEs accumulate in the mesangium and
glomerular capillary wall. The kidney is an important
regulator of AGE metabolism, and circulating low-
molecular-weight (LMW) AGE levels are elevated
in patients with diabetes and reduced GFR.82 How-
ever, the exact role of circulating LMW AGEs in the
pathogenesis of DKD is uncertain. More recently, it
was shown that serum from patients with type 2 dia-
betes and various degrees of reduced kidney function
contains high-molecular-weight ligands (.50 kDa),
mainly protein-bound N-carboxymethyllysine, that
bind and activate the receptor for AGE (RAGE).83
Various pathways downstream of RAGE have been
implicated in the development of diabetic complications.
In particular, experimental models of diabetes that over-
express RAGE show evidence of damage to kidney
function and structure.84 Unfortunately, the relationship
between serum AGE concentrations and markers of
reduced kidney function in diabetes still remains to be
clearly dened. Despite this, one study has shown that
levels of protein glycation, oxidation and nitration ad-
ducts (measured in plasma ultraltrates with a 12-kDa
cutoff, and determined by liquid chromatography
tandem mass spectrometry [LC-MS/MS]) are related to
albuminuria, but not GFR. In that study, adduct levels
were not signicantly different among plasma samples
collected from individuals with type 1 diabetes and
microalbuminuria who had stable or an early decline in
GFR decline. However, 3 protein damage adduct residues
were decreased and 3 were increased in microalbumi-
nuric compared with normoalbuminuric individuals. The
Am J Kidney Dis. 2014;63(2)(suppl 2):S39-S62
Markers of DKD Development and Progression
largest differences were of N-formylkynurenine protein
adduct residue and Nv-carboxymethylarginine (CMA)
free adduct, for which levels were substantially lower in
microalbuminuric compared with normaoalbuminuric
individuals (P , 0.001 for both).85
The potential value of urinary excretion of proteins
modied by AGEs as biomarkers for albuminuria has
been highlighted in recent studies. In participants with
both type 1 and type 2 diabetes, it has been shown
that that there is a graded relationship between level
of albuminuria and urinary AGE-modied proteins that
is independent of GFR (Fig 4). In particular, urinary
N-carboxymethyllysine excretion was found to be
increased in both micro- and macroalbuminuric partic-
ipants with type 1 diabetes compared with normoalbu-
minuric participants.86 However, it remains to be shown
that changes in urinary protein-bound AGE concentra-
tions precede an increase in urinary albumin excretion
or whether their measurement adds to the prognostic
ability of traditional DKD progression markers.
MARKERS OF INFLAMMATION AND
OXIDATIVE STRESS
Recent studies from the Joslin Diabetes Center have
examined the contribution of inammatory processes
in the progression of kidney damage in people with
diabetes. In one study, 5 inammatory markers,
interleukin 6 (IL-6), IL-8, monocyte chemoattractant
protein 1 (MCP-1), interferon ginducible protein (IP-
10), and macrophage inammatory protein 1d, were
measured in urine samples collected from individuals
with type 1 diabetes. Baseline urinary concentrations
of these inammatory markers were found to be
signicantly higher in those with an early progressive
kidney function decline (.3 mL/min/1.73 m2 per year)
compared with those who displayed stable kidney
function.87 By multivariate analysis, those with more
than 2 elevated marker levels were found to be more
than 5 times as likely to have an early progressive
decline in kidney function.
Another study investigated the relationship of 12
serum markers of inammation and apoptosis to GFR
estimated from cystatin C level in a cross-sectional
analysis of individuals with type 1 diabetes. However,
Figure 4. Urinary advanced glyca-
tion end products (AGEs) were mea-
sured in patients with (A) type 1 and
(B) type 2 diabetes, grouped according
to kidney function. Normo, Micro, and
Macro describe patients with normoal-
buminuria, microalbuminuria, and mac-
roalbuminuria, respectively. *P , 0.05
compared to Normo; #P , 0.05 com-
pared to Micro. Reproduced with
permission of Karger Publishers from
Coughlan et al.86
Am J Kidney Dis. 2014;63(2)(suppl 2):S39-S62
only levels of soluble forms of receptors for tumor
necrosis factor (TNF), for both receptor 1 and re-
ceptor 2, and Fas (which binds the Fas ligand and
results in an apoptotic response), were found to be
associated with GFR in a multivariate analysis.
Interestingly, variation in TNF receptor concentra-
tions turned out to be linked more strongly to GFR
than clinical covariates such as age and albumin
excretion.88
Subsequent studies have demonstrated the prog-
nostic signicance of baseline TNF receptor levels in
individuals with type 1 and type 2 diabetes. In a study
involving participants with type 2 diabetes, baseline
levels of TNF receptors, especially for the type 1 re-
ceptor, independently predicted progression to ESRD
over 12 years. This association was stronger in par-
ticipants without proteinuria than in those with pro-
teinuria and was independent of other markers of
the TNF pathway, markers of endothelial dysfunction,
and markers of systemic inammation, such as IL-6
and C-reactive protein (CRP). It was suggested by
the authors of that study that high circulating TNF
receptor levels could be a new predictor of ESRD
that has a more powerful predictive ability than
proteinuria.89
A further study demonstrating the potential value
of measuring circulating TNF receptor has been per-
formed in participants with type 1 diabetes with
normo- or microalbuminuria and without evidence of
hypoltration at baseline. The cumulative incidence
for reaching stage 3 CKD for individuals with the
highest TNF type 2 receptor quartile was found to be
60% after 12 years compared with only 5%-19% for
the other quartiles. In Cox proportional hazards
analysis, participants with TNF type 2 receptor values
in the highest quartile were 3-fold more likely to
experience an early decline in kidney function than
individuals in the other quartiles (HR, 3.0; 95% CI,
1.7-5.5). In this study, risk of progression to stage 3
CKD associated with high TNF type 1 receptor levels
was slightly less than that associated with high TNF
type 2 receptor levels.90
A recent report based on the DCCT/EDIC also has
suggested that high levels of inammatory markers,
S51

especially E-selectin and circulating TNF receptors 1
and 2, are important independent predictors of the
development of macroalbuminuria.91
In summary, these studies suggest that serum TNF
receptor levels are linked to the development and
progression of DKD. Circulating TNF receptor levels
may even be a stronger predictor of declining GFR
than albuminuria. However, the exact mechanisms
linking TNF receptors with DKD and whether
modulating their levels improves the prognosis of
patients with DKD remain unknown.
Experimental studies have highlighted the impor-
tance of oxidative stress, in particular the generation
of ROS, derived largely from hyperglycemia-
triggered overactivation of the NADPH (reduced
nicotinamide adenine dinucleotide phosphate) oxidase
(NOX) family of free radicalproducing enzymes
in DKD.92 Mitochondrial ROS production has been
identied as the key regulator that links the 4
major pathways implicated in the development of
hyperglycemia-driven tissue damage, in other words,
the polyol, hexosamine, PKC, and AGE pathways.
However, clinical studies have not clearly demon-
strated that markers of oxidative stress, although
elevated in individuals with diabetes compared with
those without diabetes, such as urinary 8-hydroxy-20 -
deoxyguanosine (8-OHdG) levels, offer additional
prognostic information in relation to the development
or progression of DKD over and above established
risk markers. In one study, higher, compared with
lower, urinary 8-OHdG levels were associated with
progression of albuminuria in participants with type 2
diabetes. Furthermore, multivariate logistic regres-
sion analysis suggested that urinary 8-OHdG levels
were the strongest predictor of progression of albu-
minuria among several established risk factors.93
However, another more recent study has suggested
that measuring urinary 8-OHdG did not improve the
ability to determine which patients are at risk of pro-
gressive DKD over and above measuring urinary
ACR.94
NF-E2-related factor (Nrf2) is the primary tran-
scription factor that regulates intracellular antioxi-
dants and possibly its modulation will prove to
have a renal protective effect. Unfortunately, although
experimental and some clinical studies suggested that
the Nrf2 modulator bardoxolone may have DKD
protective effects, a large clinical study involving
bardoxolone recently was ceased due to adverse
outcomes in participants randomly assigned to receive
bardoxolone.
CYTOKINES
Studies of experimental diabetes have hinted that
probrotic cytokines, such as CTGF and TGFb, may
be markers of progression of DKD.
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MacIsaac, Ekinci, and Jerums
Clinical studies evaluating the role of TGFb have
found an inconsistent link between plasma levels of
this cytokine and indexes of kidney health in people
with diabetes, which may be related to platelet
degranulation. In contrast, urinary TGFb levels
consistently have been shown to be elevated in in-
dividuals with DKD compared with those without.95
Moreover, recent studies also have reported that uri-
nary TGFb excretion is attenuated by ACE inhibition
or cholecalciferol in type 2 diabetic patients with
DKD.96
Urinary CTGF level has been found to closely
correlate with ACR. In this analysis, both parameters
were expressed on a logarithmic scale; urinary CTGF
increased 10-fold in microalbuminuric versus nor-
moalbuminuric patients and 100-fold in macro-
albuminuric versus normoalbuminuric patients. A
positive relationship between urinary TGFb and
CTGF levels also was evident.97
Larger studies have provided more evidence that
urinary excretion of CTGF is related to albuminuria
and also to GFR in type 1 diabetic patients with
diabetic nephropathy.98 In particular, one study has
shown that baseline plasma CTGF levels are associ-
ated independently with risks of ESRD and mortality
in individuals with type 1 diabetes. However, this
relationship was evident in only macroalbuminuric
individuals compared with normalbuminuric in-
dividuals. Plasma CTGF levels also were found to
signicantly improve the prediction of ESRD and
mortality for macroalbuminuric individuals in addi-
tion to conventional risk factors.99
It recently has been shown that genetic variation
in the CTGF-945 polymorphism, which has been
linked previously to susceptibility to systemic scle-
rosis, is not associated with the presence of DKD or
abnormal echocardiogram ndings after adjustment
for multiple covariates in individuals with type 2
diabetes (Fig 5).100 These ndings have been con-
rmed in another study, which interestingly showed
that CTGF-945 polymorphism was not associated
with plasma CTGF level.101 Thus, further studies are
required to assess the roles of urinary CTGF and
TGFb as markers of progression of DKD.
SERUM CYSTATIN C
An alternative approach to current methods of esti-
mating GFR is to use a different analyte to creatinine
for developing GFR predicting equations. More than
25 years ago, Grubb et al102 proposed that cystatin C
could be used as a potential marker of GFR based on
the fact that, unlike creatinine level, cystatin C levels
generally are not inuenced by extrarenal factors such
as age, sex, diet, and muscle mass. Cystatin C is an
LMW (13.3-kDa) protein that is produced at a constant
rate by all nucleated cells, freely ltered by the
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Markers of DKD Development and Progression
Figure 5. The presence of cardiac
and kidney complications in type 2 dia-
betes according to the CTGF-945 G/C
genotype: figures show the proportion
(%) of individuals with (white) and
without (black) the listed complication
according to genotype. P values for dif-
ferences in proportions are shown.
Reproduced from Patel et al.100
glomerulus, and then completely metabolized by the
proximal tubule. However, it is only recently that
studies have supported the measurement of serum
cystatin C as a simple, reliable, and accurate marker of
GFR in individuals with diabetes.103 In particular, es-
timations of GFR based on cystatin C level have been
shown to more accurately reect changes in meaured
GFR within the normal-to-high range than creatinine-
based estimates of GFR.104,105
Two recent studies also have shown that mea-
surement of serum cystatin C signicantly improves
the ability to predict which patients with diabetes are
at the greatest risk of progression to ESRD compared
with existing methods for estimating kidney function.
In one study,106 more than 1,000 patients from 3
separate cohorts with both type 1 and type 2 diabetes
and CKD stages 1-3, based on the CKD-EPI (CKD
Epidemiology Collaboration) creatinine equation,
were followed up for 8-10 years for the onset of
ESRD, which occurred in 364 participants. CKD
staging also was performed using an eGFR formula
based on cystatin C level and was found to signi-
cantly improve ESRD risk statication. Concurrent
CKD staging was seen in 62% of participants. How-
ever, participants given a higher (more advanced)
CKD stage by cystatin C versus creatinine-based
eGFR had a signicantly higher risk of ESRD than
those with concordant staging in all 3 cohorts (HR,
2.3; 95% CI, 1.8-3.1). Similarly, patients at a lower
(less advanced) CKD stage by cystatin C versus
Am J Kidney Dis. 2014;63(2)(suppl 2):S39-S62
creatinine-based eGFR had a lower risk than those
with concordant staging (HR, 0.30; 95% CI, 0.13-
0.68).106
In another study,107 serum cystatin C levels were
found to be a better predictor of ESRD than directly
measured GFR (iothalamate clearance) or serum
creatinine level. In that study, 234 Pima Indians were
followed up for a median of 10.7 years, with 68
(29%) developing ESRD. In analyses adjusted for
age, sex, diabetes duration, height, weight, HbA1c
level, and ACR, the reciprocal of serum cystatin C had
the highest area under the receiver operating charac-
teristic curve (AUROC) of 0.845 6 0.026. Models
with measured GFR or the reciprocal of serum creati-
nine had similar AUROCs, which were lower than the
model with the reciprocal of cystatin C alone (P 5 0.02
and P 5 0.03, respectively). It still is unclear whether
the superior predictive value of serum cystatin C levels
for ESRD in patients with diabetes is due to an
enhanced ability to reect true GFR beyond directly
measured GFR or its abilty to incorporate nonrenal
inuences on progressive kidney failure.107
In summary, recent work suggests that measure-
ment of serum cystatin C provides a simple and ac-
curate method for detecting early decline in kidney
function and subsequent trends in kidney function for
individuals with diabetes. Evidence also is starting to
emerge to suggest that in comparision to creatinine,
measurement of cystatin C can strengthen the asso-
ciation between eGFR and risk of death and ESRD.
S53

Although extrarenal factors may inuence cystatin
C levels, the inuence of these factors on cystatin
C levels is likely to be much smaller than their cor-
responding effects on creatinine levels. However,
although cystatin C is emerging as a very promising
alternative to creatinine-based methods for estimating
GFR, further studies are needed to investigate the
inuence of nonrenal factors on cystatin C levels.
Previous problems with cystatin C assays, including a
drift in calibration of the measurement procedures and
lack of standardization against against an international
reference, have recently been addressed, which
should help to clarify the role of cystatin C as a
marker of GFR. It also is necessary to develop
reference ranges for cystatin C levels in large numbers
of individuals with diabetes across a wide range of
kidney function. Given these limitations, together
with the added expense of measuring cystatin C
compared to creatinine and the lack of access to
laboratories that routinely measure cystatin C,
creatinine-based methods will almost cetainly remain
the most clinically applicable way of estimating GFR
in the immediate future.
TUBULAR MARKERS
During the last 10 years, it has been shown that
tubulointerstitial, in addition to glomerular, damage is
an important factor in the progression of DKD.108 The
urinary proximal tubular markers that have been
studied most vigorously include kidney injury mole-
cule 1 (KIM-1), neutrophil gelatinase associated lip-
ocalin (NGAL), and liver-type fatty acid-binding
protein (L-FABP).
One study has shown that higher baseline levels of
KIM-1 and NGAL, but not L-FABP, are associated
with a faster decline in GFR in proteinuric patients
with type 1 diabetes. However, after accounting for
known progression promoters of DKD such as albu-
minuria, diabetes duration, glycemic control, and BP,
this relationship was found to lose signicance.109
Similarly, in a 4-year follow-up study of participants
with type 2 diabetes but with the majority having
eGFRs $ 60 mL/min/1.73 m2 and normoalbumi-
nuria, higher urinary baseline levels of the tubular
biomarkers KIM-1 and glycoprotein nonmetastatic
melanoma B (Gpnmb) were found to be associated
with a faster decline in eGFR in univariate analysis.
However, this association was no longer signicant
after adjusting for known risk factors, such as baseline
albuminuria, BP, eGFR, or HbA1c level.110
Despite this, urinary L-FABP levels have been
shown to be associated with progression of DKD,
dened as progressive albuminuria or the develop-
ment of ESRD. For individuals with type 2 diabetes
who were followed up for 4 years and who had an
initial eGFR . 60 mL/min/1.73 m2, high urinary
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MacIsaac, Ekinci, and Jerums
L-FABP levels were found to be a strong and inde-
pendent predictor of progressive DKD.111 In a cross-
sectional study of participants with type 2 diabetes,
heart fatty acid-binding protein (H-FABP), a marker
of distal tubular damage, was the only tubular marker
associated with eGFR after adjustment for other risk
markers of progressive DKD.112
An inconsistent relationship between urinary
tubular markers and regression or progression of
albuminuria has been reported. One study involving
participants with type 1 diabetes showed that regres-
sion of microalbuminuria was associated with lower,
compared to higher, urinary levels of both KIM-1 and
NGAL, with this relationship being independent of
other clinical or biochemical variables. In contrast,
another study involving participants with type 2 dia-
betes did not nd a relationship between levels of
tubular biomarkers and progression from normo- to
microalbuminuria or regression of micro- to normoal-
buminuria.110 As mentioned, increased urinary levels
of L-FABP have been linked to progressive albumin-
uria. One study has shown that urinary L-FABP levels
independently predict the development of micro-
albumuria in individuals with type 1 diabetes.113
As mentioned in the previous section, cystatin C
usually is freely ltered by the glomerulus and then
completely reabsorbed and metabolized by the prox-
imal tubule. Therefore, the appearance of cystatin in
urine increasingly is becoming recognized as a marker
of tubular dysfunction. The urinary cystatin C to
creatinine ratio has been shown to independently
predict the development of CKD stage 3 in in-
dividuals with type 2 diabetes after adjustment
for a number of covariates. However, in a separate
analysis of individuals who initially had eGFRs
. 60 mL/min/1.73 m2 and normoalbuminuria, uri-
nary cystatin C to creatinine ratio was not associated
independently with the rate of decline in eGFR.114
In summary, although higher levels of tubular injury
markers may be associated with a faster decline in GFR
and progression or remission of albuminuria in some
studies, it has been difcult to show that measurement
of tubular markers offers additional prognostic infor-
mation over and above established risk markers.
Members of the FABP family are emerging as the
tubular markers with the greatest chance of offering
added predictive value for progressive DKD over and
above that offered by established risk markers.
URIC ACID
During the last 5 years, evidence has accumulated
to suggest that increased levels of serum uric acid are
an independent risk factor for progressive DKD.
Interpreting studies that have attempted to link
elevated serum uric acid levels with DKD has been
complicated by the confounding inuences of GFR
Am J Kidney Dis. 2014;63(2)(suppl 2):S39-S62
Markers of DKD Development and Progression
and use of diuretics, which can alter uric acid levels.
Despite this, many observational studies have shown
a statistically independent association between uric
acid level and reduced GFR or the development of
proteinuria.
In a cross-sectional study of participants with type
1 diabetes who had normo- or microalbuminuria, uric
acid levels in the high-normal range were found to be
associated independently with reduced GFR as
measured by cystatin C level.115 In another study, the
same group of researchers were able to demonstrate
an independent dose relationship between baseline
uric acid levels and risk of early GFR loss and risk of
developing CKD stage 3 in participants with type 1
diabetes who were followed up for 4-6 years. Inter-
estingly, uric acid levels were not associated with risk
of progression or regression of albuminuria in that
study.116
Studies demonstrating a relationship between
elevated uric acid levels and the development of
microalbuminuria have remained elusive. In a study
from the Steno Diabetes Centre, baseline uric acid
levels were predictive of the development of macro- but
not microalbuminuria in an inception cohort study of
263 participants with type 1 diabetes followed up for
18 years.117 In that study, the HR for the development
of macroalbuminuria was 2.37 (95% CI, 1.04-5.37) for
every 100-mmol/L increase in serum uric acid level
(P 5 0.04). In another study involving participants
with type 1 diabetes, baseline uric acid levels were
associated with the development of albuminuria at a
6-year follow-up visit in an analysis that examined
the risk of developing micro- or macroalbuminuria.118
In multivariate analysis, each 60-mmol/L increase in
serum uric acid level at baseline was associated with
an 80% increased risk for developing albuminuria.
For individuals with type 2 diabetes, normal kidney
function, and without overt proteinuria, baseline uric
acid levels were associated with the development of
CKD (dened as overt proteinuria or eGFR , 60 mL/
min/1.73 m2) over a 5-year follow-up. After adjusting
for multiple covariates, each 1standard deviation
increase in uric acid levels was associated with a 21%
increased risk of CKD.119 Furthermore, post hoc
analysis of the RENAAL trial has demonstrated that
the degree of serum uric acid reduction at 6 months
posttreatment with losartan is linked to a lower risk of
doubling of serum creatinine level or the development
of ESRD in proteinuric individuals with type 2 dia-
betes.120 This relationship also was shown to be in-
dependent of other risk markers, such as eGFR and
albuminuria.
Although the exact mechanisms linking elevated
uric acid levels with DKD are not fully understood,
contributions from inammatory pathways, endothe-
lial dysfunction, and hypertension most likely play a
Am J Kidney Dis. 2014;63(2)(suppl 2):S39-S62
role. To date, no clinical study has clearly shown that
serum uric acid levels start to increase before GFR
declines in individuals with diabetes who are destined
to develop DKD. This area of research also awaits
results of clinical studies examining the impact of
lowering serum uric acid levels to indexes of kidney
health in people with diabetes.121
PROTEOMICS
Recently, techniques of urinary proteome analysis,
which have taken a systematic approach to the iden-
tication and quantication of urinary proteins, have
been used in an attempt to identify novel urinary
markers for the development and progression of
DKD. In particular, the prole of the LMW protein
fraction (,2,000 Da) in urine, which is composed of
intact LMW proteins and peptide fragments, has been
examined.
A sophisticated analysis of the urinary peptidome
suggested that it may be possible to identify which
patients with microalbuminuria and type 1 diabetes
are at greatest risk of experiencing an early decline in
kidney function. Of the peptides identied by liquid
chromatography matrix-assisted laser desorption/
ionization time-of-ight mass spectrometry, 3 were
present at lower levels in urine of patients with early
kidney function decline (fragments of a-1(IV)
collagen, a-1(V) collagens, and tenascin-X) and 3
were increased (fragments of inositol pentaki-
sphosphate 2-kinase, zona occludens 3, and FAT
tumor suppressor 2) compared with individuals with
stable kidney function.122
A large cross-sectional study has identied pepti-
dome patterns that have differentiated patients with or
without diabetes, even in the absence of DKD, using
capillary electrophoresis coupled with mass spec-
trometry. Furthermore, urinary biomarkers also could
distinguish individuals with type 1 diabetes from
those with type 2 diabetes. Specic collagen frag-
ments were associated with diabetes and type of
diabetes, suggesting that collagen turnover and
extracellular matrix changes are indicative of the
molecular pathophysiology of diabetes. The most
pronounced diabetes-associated urinary proteome
changes were a signicant reduction in specic
collagen a-1 (I) and (III) fragments, with these
changes being signicantly dramatic in individuals
with type 2 diabetes than in those with type 1 dia-
betes. Additional biomarkers that were identied
included those implicated in inammatory and pro-
thrombotic processes.123
Using this methodology, 2 further studies have
investigated the prognostic signicance of the so-
called CKD biomarker classier (CKD273) to iden-
tify patients at risk of progressive DKD. This urinary
peptide classier, consisting of 273 dened urinary
S55

Figure 6. Time course of the CKD273 classifier and albumin
excretion rate (AER) in individuals with type 1 or type 2 diabetes
who either progressed (grey line) or did not progress (black line)
to develop macroalbuminuria. The dotted line depicts the cutoff
of 0.343 arbitrary units for the CKD273 classifier, the score
that previously has been shown to discriminate between
CKD273 casepositive or negative individuals, and 20 mg/min
for AER, the accepted threshold for the development of microal-
buminuria. Reproduced with permission of the American Dia-
betes Association from Zurbig et al.125
peptides, previously has been shown to be a good
classier of individuals with and without CKD
regardless of cause.124 In a longitudinal study of 16
participants with type 1 or 2 diabetes who all devel-
oped macroalbuminuria, the transition from normo- to
microalbuminuria occurred 3.4 6 2.1 years before
progression from micro- to macroalbuminuria. In
contrast, a CKD273 classier-positive pattern was
detected at 4.9 6 2.2 years (P 5 0.016) before the
transition from micro- to macroalbuminuria (Fig 6).
Discrimination between a cutoff score for being
CKD273 classierpositive or negative therefore of matrix proteins within the mesangium, such as type
was identied at a time 1.5 years before the devel-
opment of microalbuminuria in participants who were
destined to develop macroalbuminuria. This suggests
that patients with normoalbuminuria and a CKD273
classierpositive pattern are at increased risk for the
development of macroalbuminria (overt diabetic ne-
phropathy) and can be identied as such before the
development of microalbuminuria. In urine of patients
with both type 1 and type 2 diabetes, fragments of
collagen were detected in lower levels for those who
developed macroalbuminuria compared with those
who did not.125
Furthermore, CKD273 classier score has been
shown to be associated independently with progres-
sion of albuminuria in a case-control study involving
participants with type 2 diabetes. In that study, cases
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MacIsaac, Ekinci, and Jerums
were dened by transition from normo- to micro-
albuminuria or from micro- to macroalbuminuria
during a 3-year follow-up, whereas controls who were
age-, sex-, and albuminuria statusmatched did not
display a transition in albuminuria status. The
CKD273 classier predicted the development and
progression of albuminuria (odds ratio, 1.35; 95% CI,
1.02-1.79) even after accounting for baseline albu-
minuria and eGFR. A signicant negative correlation
also was seen between CKD273 classier score and
changes in eGFR between follow-up and baseline. In
a fashion similar to the other studies described, frag-
ments of collagen showed signicantly different
expression between cases and controls.126
Importantly, these results suggest that the CKD273
classierpositive pattern may help identify normo-
albuminuric patients who are at risk of progressive
DKD. It is not yet clear whether a capillary elec-
trophoresis coupled with mass spectrometry pattern,
attributed largely to collagen fragments, or whether
novel biomarkers, identied by capillary electropho-
resis coupled with mass spectrometry technology, will
be the most appropriate path toward earlier diagnosis
of DKD.
OTHER NOVEL MARKERS
Fibroblast growth factor 23 (FGF-23) has been
implicated as a risk marker for progressive CKD.
Serum FGF-23 levels have been shown to be an in-
dependent predictor of an outcome comprising death,
doubling of serum creatinine level, and/or dialysis in a
small study of 55 participants with type 2 diabetes and
macroalbuminuria who were followed up for 30
months. FGF-23 is emerging as an important regu-
lator of phosphate metabolism, but interestingly, in
the mentioned study, FGF-23 levels were not asso-
ciated with levels of markers of bone metabolism or
phosphate.127
DKD usually is associated with the accumulation
IV collagen. One study has shown that high urinary
excretion of type IV collagen is associated indepen-
dently with an annual decline in eGFR over an 8-year
observational period.128 A relationship between uri-
nary type IV collagen and eGFR decline also was
seen in normoalbuminuric individuals. Measurement
of urinary type IV collagen therefore may prove to be
useful in identifying people at risk of a progressive
decline in GFR in the absence of increasing albu-
minuria. How these ndings of increased full-length
collagen urinary excretion relate to reports of
reduced collagen fragments, detected in proteomic
studies, awaits further investigation.
Quantifying podocyte injury through the measure-
ment of urinary markers has been proposed as a novel
way of identifying people at risk of progressive DKD.
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Markers of DKD Development and Progression

Table 1. Summary of the Usefulness of Various Markers for the Development and Progression of DKD

Marker for DKD Advantages Disadvantages

Albuminuria Urinary albumin levels within the microalbuminuric
range predict ESRD
GFR Best measure of kidney function
Glucose An important marker because hyperglycemia is the
initiator of DKD
Blood pressure An important promoter of DKD; also important in CV
risk reduction
Lipids Important in CV risk reduction; lipid-modifying
agents may have renal-protective effects
independent of changes in lipid profile
Soluble TNF Circulating levels of TNF receptors have been
receptors shown to predict ESRD and possibly have a more
powerful predictive ability than proteinuria
Uric acid Easy to measure; levels also may relate to CV risk
Tubular markers Easy to measure in urine sample
Urinary proteome The appearance of the CKD273 biomarker
classifier is an earlier marker for the risk of the
development of proteinuria, even prior to the
onset of microalbuminuria
Serum cystatin C Predicts ESRD better than creatinine-based eGFR
methods and possibly directly measured GFR
Abbreviations: AER, albumin excretion rate; CKD, chronic kidney disease; CV, cardiovascular; DKD, diabetic kidney disease;
(e)GFR, (estimated) glomerular filtration rate; ESRD, end-stage renal disease; TNF, tumor necrosis factor.
High variability, low specificity for DKD; spontaneous
regression and DAER within the microalbuminuric range
s DGFR
Routine methods for accurately estimating GFR in the
normal-high range are still lacking
Targets still to be optimally defined; evidence documenting
that intensive glycemic control prevents ESRD is sparse
Targets are still to be optimally defined
The relationship between components of the lipid profile
and risk for DKD progression is not optimally defined
The relationship between TNF receptors and ESRD
remains to be confirmed in various patient populations
attending different centers
Kidney disease outcome intervention studies to target uric
acid levels are still required
The prognostic significance of their measurement over
and above established risk factors remains to be fully
defined
Clinical assays are lacking
Expensive and the standardization of assays is not yet
universal

Approaches have included measuring urinary excre-
tion of podocytes and the quantication of podocyte-
associated molecules by measurement of mRNA
proles of synaptopodin, podocalyxin, CD2-AP,
a-actinin-4, and podocin.129,130 However, to date,
quantication of podocyte injury, although strongly
linked to the severity of albuminuria, has not been
demonstrated to be an independent marker of GFR loss.
Another approach that warrants further investiga-
tion is the measurement of circulating and urinary
microRNAs (miRNAs) and the usefulness of RNA-
based therapies. miRNAs are small noncoding
RNAs that control gene/protein expression through
degrading and/or inhibiting mRNA and hence protein
synthesis. Currently, clinical studies investigating the
usefulness of miRNAs as biomarkers for the devel-
opment and progression of DKD are lacking. How-
ever, experimental evidence suggests that inhibiting
miRNA-192, which is upregulated in mesangial
cells and glomeruli by TGFb in diabetic animals, can
ameliorate renal brosis and proteinuria.131
SUMMARY
It remains difcult to determine which patients with
diabetes will develop DKD and progress to a state of
declining GFR and the development of ESRD. The
search continues for biomarkers that assist with risk
stratication of patients with diabetes over and
beyond that conferred by established risk markers for
DKD (Table 1). Research in this area is hampered
because there still is no accepted gold standard for the
diagnosis and progression of DKD. There is a need to
develop better early risk markers for DKD in clinical
practice and clinical trials. Currently, reducing the
risk of doubling of serum creatinine level, develop-
ment of ESRD, and death remain the standard against
which therapeutic interventions to slow the progres-
sion of DKD ultimately are judged.
Albuminuria has been the traditional parameter on
which the diagnosis and progression of DKD has
been based. However, albuminuria, especially within
the microalbuminuric range, although remaining an
important risk marker, lacks the specicity and
sensitivity to optimize the risk stratication of patients
at risk for the development and progression of DKD.
Recently, more emphasis has been placed on changes
in GFR, especially an early decline in GFR before a
threshold of 60 mL/min/1.73 m2 is reached. However,
to optimize this approach in clinical practice,
improvement in existing methods for routinely esti-
mating GFR in the normal-to-high range will be
required.

Am J Kidney Dis. 2014;63(2)(suppl 2):S39-S62 S57


Apart from albuminuria and GFR, serum uric acid
levels and measurement of serum levels of soluble
TNF receptors (both type 1 and type 2) appear to be
the most promising biomarkers for DKD, especially
in relation to changes in GFR. Measurement of the
urinary peptidome and resultant identication of the
CKD273 peptide classier have been shown to be
promising biomarkers for patients with diabetes at
risk for the development of proteinuria. However, this
approach awaits the development of a clinical assay
and further studies to dene the prognostic signi-
cance of this marker, especially in relation to risk for
declining GFR.
While waiting for the development and appraisal of
new risk markers for DKD, greater attention to family
history, smoking history, absolute (not categorical)
AER values, precise GFR measurements, glycemic
control, ambulatory BP measurements, and plasma
lipid levels may enhance predictive accuracy for
detecting people at greatest risk for the development
and progression of DKD.
ACKNOWLEDGEMENTS
Support: The development of this journal supplement was
funded by Novo Nordisk. The authors work described in this
manuscript was supported by a Regional Diabetes Support grant
from Novo Nordisk and the St Vincents Hospital, Melbourne
Research Endowment Fund. No other honoraria or remuneration
were received for this work. Technical editing was provided by
Watermeadow Medical, funded by Novo Nordisk. Costs associ-
ated with publication were funded by Novo Nordisk.
Financial Disclosure: Prof MacIsaac has received honoraria
for lectures and travel support from Eli Lilly, Novo Nordisk,
Sano Aventis, Astra Zeneca, Merck Sharp & Dohme, Servier,
Boehringer Ingleheim, and Novartis and has received research
grants from Novo Nordisk. Dr Ekinci has received honoraria for
lectures from Eli Lilly, Novo Nordisk, and Astra Zeneca. Prof
Jerums declares that he has no relevant nancial interests.
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