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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-


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

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MacIsaac, Ekinci, and Jerums

certain circumstances, an increase in serum creatinine threshold of 60 mL/min/1.73 m2, to identify those at
level and hence a decrease in estimated GFR (eGFR) risk of progressing to advanced kidney disease. In
may represent transient changes in kidney perfusion order to maximize the utility of this strategy, methods
or function that are not necessarily related to the for estimating GFR in the high-normal range will
causal pathway that is driving the development of need to be improved.
DKD. From a practical point of view, hard renal end Metabolic abnormalities associated with DKD in-
points usually are worth considering only in large clude hyperglycemia, hypertension, and dyslipidemia.
long-term studies involving high-risk patients with There appears to be a hemoglobin A1c (HbA1c)
sufcient events occurring to allow meaningful sta- threshold for the development of reduced kidney
tistical comparisons to be made. function of w6.5% (w48 mmol/mol). Although
Apart from albuminuria and GFR, established or glycemic targets should be individualized, intensive
potential risk markers for DKD include hyperglyce- glycemic control clearly has been shown to reduce the
mia, hypertension, circulating TNF receptors, hyper- incidence of macro- and microalbuminuria, and recent
uricemia, tubular injury markers, presence of specic evidence indicates that very intensive glycemic con-
urinary peptide classiers, and serum cystatin C level. trol may slow the decline in GFR and possibly the
However, each of these has limitations and we remain progression to ESRD.
in need of reliable risk biomarkers to determine which There also is a recognized role for hypertension in
individuals are at risk of developing DKD that will accelerating DKD progression, and lowering BP can
progress to ESRD. slow the deterioration in kidney function. In obser-
The cause of DKD includes irreversible (age, sex, vational studies, a continuous relationship between
ethnicity, family history, and duration of diabetes) BP level and kidney and cardiovascular events has
and modiable (hyperglycemia, hypertension, albu- emerged in people with diabetes, although recent
minuria, dyslipidemia, and smoking) risk factors. intensive BP-lowering trials have failed to dene the
Despite evidence of genetic susceptibility and fa- optimal BP targets for slowing the progression of
milial aggregation, associations between particular kidney and cardiovascular disease. Disturbances in
gene(s) and indexes of DKD have yet to be reliably circadian BP variability are associated with a number
identied. Recent studies have linked specic ge- of adverse kidney disease outcomes and may predict
netic variants with different DKD phenotypes, but the development of microalbuminuria, DKD pro-
candidate gene studies and genome-wide linkage gression, and/or mortality risk.
studies have failed to identify a major gene respon- The role of dyslipidemia remains poorly dened,
sible for familial clustering in type 1 and type 2 although higher levels of LDL (low-density lipopro-
diabetes, and prospective studies are needed to tein) cholesterol and triglycerides and lower HDL
investigate which epigenetic factors predispose to (high-density lipoprotein) cholesterol concentrations
DKD. Once the DKD disease process has started, appear to be associated with greater risks of albu-
metabolic, inammatory, and hemodynamic path- minuria and declining GFR, and improvement in
ways promote progression, sharing a nal common these parameters may reduce albuminuria. Statins and
manifestation of excess accumulated connective tis- fenobrate may have a renal preservation effect.
sue, renal brosis, and/or scarring. Several markers of renal structure and function
Albuminuria has long been used to monitor the have been linked to the development and progres-
onset and progression of DKD, but microalbuminuria, sion of DKD. The relationship between structural
historically considered a strong predictor of progres- changes and functional DKD progression is not
sion to proteinuria, recently has been observed to clear cut, and biopsy ndings currently are not
follow a remission/regression trajectory of less certain used to determine disease progression. However,
prognostic value. Furthermore, microalbuminuria is markers of renal vascular function may provide
neither a universal nor a sensitive or specic marker useful prognostic information. For example, increased
for progressive DKD, and recent studies have chal- intrarenal vascular resistance and indexes of extra-
lenged the previously accepted chronological rela- renal vascular dysfunction, such as abnormal echo-
tionship between albuminuria and GFR decline. cardiogram ndings, are associated with reduced
In type 1 diabetes, early accelerated loss of GFR kidney function. Endothelial dysfunction also is
appears to be a useful predictor of ESRD, whereas in implicated in the progression of DKD, and asymmet-
type 2 disease, decline in GFR often predates mac- rical dimethylarginine (ADMA), the major endoge-
roalbuminuria (although a decline predictive of nous inhibitor of nitric oxide production, recently has
ESRD typically depends on progression to macro- emerged as a novel and important predictor of DKD
albuminuria). Given this relationship between albu- progression.
minuria and GFR, there now is a move toward using A number of urinary and serum markers have been
both, particularly an early decline in GFR above a proposed as risk biomarkers for DKD progression.

S40 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 normoalbuminuric patients with diabetes who are at
receptors seem to be the most promising. Many ob- risk of progressive DKD.
servational studies have shown a statistically inde- Other potential signiers of DKD risk and/or pro-
pendent association between serum uric acid level and gression require further evidence-based support
reduced GFR or the development of proteinuria, before becoming reliable disease markers. Candidates
although a relationship between elevated uric acid here include urinary protein-bound advanced glyca-
levels and microalbuminuria is yet to be determined. tion end products, certain markers of oxidative stress,
Support for a prognostic role for uric acid level will probrotic cytokines such as connective tissue growth
require evidence that elevations in uric acid levels factor (CTGF) and transforming growth factor b
precede a decline in GFR in at-risk patients and (TGFb), cystatin C, and tubular markers in the fatty-
clarication of the effects of lowering uric acid levels acid binding protein family. Novel markers under
in individuals with diabetes without kidney disease. investigation include broblast growth factor 23
Current evidence suggests that serum levels of TNF (FGF-23), urinary type IV collagen, and circulating
receptors (types 1 and 2) may independently predict and urinary microRNAs.
progression to ESRD, possibly even with greater While awaiting further studies of potential risk
accuracy than albuminuria, although the precise biomarkers for DKD, risk prediction should continue
mechanisms remain unknown, as does the effect of to focus on family history, smoking history, absolute
modulating these receptors. Measurement of the uri- (as opposed to categorical) albumin excretion rate,
nary peptidome and specically the CKD273 peptide precise GFR measurements, glycemic control, ambu-
classier also shows potential as a biomarker in latory BP measurements, and plasma lipid levels.

INTRODUCTION population-based cohort (1,268,029 participants)


with measures of estimated GFR (eGFR) and pro-
It is now well recognized that the incidence of
teinuria who were followed up for 4 years showed
diabetes is increasing worldwide, mainly due to the
that participants with diabetes and proteinuric chronic
dramatic increase in type 2 diabetes. The public health
kidney disease (CKD), with no history of a previous
impact of this epidemic is vast because diabetes now
myocardial infarction, have incident myocardial
is the leading cause of end-stage renal disease (ESRD)
infarction rates that are greater than those for par-
in Western countries. Traditionally, diabetic kidney
ticipants with a previous myocardial infarction.4
disease (DKD) has been referred to as diabetic ne-
Microalbuminuria is an early step during the pro-
phropathy, dened as persistent clinically detectable
gressive increase in albumin excretion rates (AERs)
proteinuria linked to elevated blood pressure (BP) and
that typically characterizes DKD. This subclinical
decreased glomerular ltration rate (GFR). Diabetic
increase in urinary albumin excretion corresponds to
nephropathy has been reported to occur in 25%-40%
an AER of 20-200 mg/min (30-300 mg/d) or an
of people with type 1 or type 2 diabetes. People with
albumin-creatinine ratio (ACR) of 2.5-35 mg/mmol in
DKD are not only at signicant risk of progression to
males and 3.5-35 mg/mmol in females. The appear-
ESRD, there also is a concomitant increase in car-
ance of microalbuminuria usually is regarded as
diovascular morbidity and mortality. Thus, it is of
incipient nephropathy, but it also is a risk factor for
critical importance to identify people at greatest risk
both micro- and macrovascular disease in people with
and initiate kidney- and cardiovascular-protective
diabetes, as mentioned. Recently, work has suggested
treatments as early as possible.
that some individuals with diabetes and decreased
This concept has been highlighted in recent studies
GFR may not have an increased urinary AER.5 It
that have shown a strong and graded relationship
therefore is timely that the contemporary approach to
between indexes of kidney dysfunction and inc-
staging DKD now is based on both level of urinary
reased risk for cardiovascular events and all-cause
albumin excretion and eGFR.6
mortality in people with diabetes. Studies involving
Both absolute change and rate of change in albu-
2 separate cohorts of individuals with type 1 diabetes
minuria are linked strongly to the development and
have indicated that the excess mortality seen in
progression of reduced kidney function in diabetes.
people with type 1 diabetes is accounted for almost
Despite this, the sensitivity and specicity of albu-
entirely by those with DKD.1,2 An analysis of ran-
minuria as a marker of progressive DKD recently has
domized controlled trials involving participants with
been challenged, with more emphasis being placed on
type 2 diabetes also found a 29-fold difference in
early changes in GFR.7 The natural history of DKD in
annualized mortality rates, with the highest mor-
terms of changes in GFR is shown in Fig 1. Although
tality rates reported from trials involving individuals
the state of hyperltration has been shown to predict
with DKD.3 Furthermore, a recent report of a large

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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
Figure 1. Clinical patterns of diabetic kidney disease.
(A) Normo-hypofiltration; (B) normo-hyper-normo-hypofiltration;
viewed as a promoter of DKD in type 1 diabetes, that
(C) normo-hyper-normofiltration; and (D) persistent normofiltra- is, it usually only develops after the onset of reduced
tion. Abbreviation: GFR, glomerular filtration rate. kidney function. However, in type 2 diabetes, it most
likely is an important initiator and promoter of DKD.
Albuminuria, dyslipidemia, and smoking are other
the progression of albuminuria in many, but not all, well-recognized modiable initiators and promoters
studies, the concept that hyperltration predisposes of DKD.
to the development of CKD stage 3, that is, a re- The pathophysiology of DKD is characterized
duction in GFR to ,60 mL/min/1.73 m2, remains to by the activation of metabolic, inammatory, and
be proved.8 hemodynamic pathways (Fig 2). Metabolic pathways
Recently, the prognostic signicance of identi- primarily result from chronic hyperglycemia that
fying individuals with diabetes who have an early leads to increased protein kinase C (PKC) activity,
(starting at ,60 mL/min/1.73 m2) decline in GFR abnormalities in polyol metabolism, heightened sec-
(.3.5 mL/min/1.73 m2 per year) that is over and retion of probrotic cytokines (such as transforming
above what would be expected with aging alone has growth factor b [TGFb]) and connective tissue
been highlighted, with this early decline being linked to growth factor [CTGF]), nonenzymatic glycosylation
the development of ESRD in type 1 diabetes.9 Apart that causes glycation of glomerular and tubular pro-
from this, established risk factors for DKD are hyper- teins, and the generation of AGEs. From this, reactive
glycemia, hypertension, duration of diabetes, age, sex, oxygen species (ROS) are produced, and they,
smoking, and family history. Emerging risk factors for together with an inammatory process, play a key role
the development and progression of DKD include in the development of DKD.11,12 The hemodynamic
markers of oxidation and inammation, probrotic pathways underlying the development of DKD cause
cytokines, uric acid level, advanced glycation end systemic and intraglomerular hypertension. They
products (AGEs), levels of markers (both functional arise by the activation of vasoactive systems such as
and structural) of vascular dysfunction, kidney struc-
tural changes, and tubular biomarker levels.
This review summarizes the prognostic signicance Box 1. Initiators and Promoters of DKD
of established and novel markers that have been
Initiators of DKD
linked to the development and progression of DKD  Hyperglycemia
in epidemiologic, experimental, and clinical studies.  Predisposing genesa
The potential impact of modulating these markers also
Promoters of DKD
is examined. Despite multifactorial strategies that  Hyperglycemia
target existing risk factors, many individuals with  Hypertension
DKD progress to ESRD. Identifying novel factors  Dyslipidemia
associated with DKD progression may result in  Insulin resistance
better understanding of the development and progres-  Smoking
 Procoagulant state
sion of this important diabetes-related complication.  Long duration of diabetesa
Furthermore, it also may result in the development of  Anemia
new interventions to prevent or alleviate decreased  Ethnicity/westernizationa
kidney function in people with diabetes.  Sexa
 Agea
INITIATORS AND PROMOTERS OF DKD  Albuminuria

There are several potential initiators and promoters Abbreviation: DKD, diabetic kidney disease.
a
of DKD, both irreversible and reversible (Box 1). The Irreversible risk factors.

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

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MacIsaac, Ekinci, and Jerums

glycemia ameliorates a decline in GFR and may even times that some evidence has emerged to suggest that
delay the progression to ESRD. very intensive glycemic control can slow GFR loss
A recent study from the DCCT/EDIC (Epidemi- and possibly the progression to ESRD.
ology of Diabetes Interventions and Complications)
Research Group has shown that long-term risk for Genetic Markers
the development of GFR , 60 mL/min/1.73 m2 was Genetic and epigenetic factors may inuence the
signicantly lower for people who initially were development and progression of DKD. Although
treated to achieve tight glycemic control.17 A sub- there is evidence for genetic susceptibility for the
study from ADVANCE has shown that individuals development of DKD, identication of causative
with type 2 diabetes randomly assigned to tight, genes has proved to be elusive. Familial aggregation
compared with conventional, glycemic control have a of DKD is a well-recognized phenomenon. Diabetic
reduced risk of developing ESRD.20 Furthermore, siblings of patients with ESRD due to diabetes are
preliminary results from the Joslin Diabetes Center known to be at 5-fold higher risk of ESRD compared
suggest that sustained improvement in glucose control with those without a family history.24,25 Despite this
in patients with type 1 diabetes with overt proteinuria recognized association, many of these high-risk in-
can reduce the long-term risk of ESRD. A 1% dividuals are not being targeted for early and inten-
improvement in HbA1c level over a follow-up of sive risk-factor modication. One recent study has
approximately 3.5 years has been reported to be shown that diabetic siblings of patients with ESRD
associated with a hazard ratio (HR) for ESRD of 0.72 due to diabetes have a high frequency of albuminuria
(95% CI, 0.61-0.89), which was independent of other (46%), suboptimal BP control (65%), suboptimal
covariates.21 glycemic control (HbA1c . 7.0%; 43%), smoking
A meta-analysis of intensive glycemic control (26%), and failure to receive RAAS-modifying agents
studies in type 2 diabetes has shown that compared (42%).26
to conventional control, intensive glycemic control Although several candidate DKD genes have been
reduces the risk for the development of micro- identied, the association between a particular gene
albuminuria (risk ratio, 0.86; 95% CI, 0.76-0.96) and and indexes of diabetic kidney dysfunction have
macroalbuminuria (risk ratio, 0.74; 95% CI, 0.65- not been vigorously replicated. Some of the candi-
0.85). Although risk for the development of ESRD date genes for DKD are listed in Box 2. Of all
was reduced by 31% (risk ratio, 0.69; 95% CI, the candidate genes, those related to RAAS regula-
0.45-1.05), this reduction with intensive glucose tion have been studied most extensively. A meta-
lowering was not signicantly different compared with analysis of 12 studies examined the relationship
conventional glucose control. This result possibly was between angiotensin-converting enzyme (ACE) inser-
related to the relatively low incidence of ESRD tion/deletion gene polymorphisms and ESRD risk in
(,1.5%) compared with those of microalbuminuria patients with DKD. A signicant association was
(23%) and macroalbuminuria (5%).22 found between the deletion allele or homozyg-
Some of the most impressive information sup- ous deletion genotype and ESRD susceptibility. Of
porting the benecial effects of tight glycemic control note, these associations were found in the overall
on the kidneys of people with diabetes has come from
a study involving individuals with DKD who received Box 2. Candidate Genes Implicated in the Susceptibility for the
a pancreatic transplant. After 5 years of normogly- Development of DKD
cemia, achieved by pancreatic transplantation, biopsy  Angiotensin-converting enzyme (ACE)
specimens of transplant recipients native kidneys  Angiotensinogen
showed no change in kidney structure. However, after  Angiotensin II receptor (type 1)
10 years of normoglycemia, remarkable remodeling  Aldose reductase
 Apolipoprotein E
of kidney structure with near-complete reversal of  Atrial natriuretic peptide
many of the structural parameters associated with  Heparin sulfate
classic diabetic nephropathy was observed.23  Intercellular adhesion molecule 1 (ICAM)
The HbA1c threshold for the development of  Matrix metalloproteinase
reduced kidney function remains to be clearly dened,  Methylene metalloproteinase 9 (MM-9)
 Na/H exchanger
but possibly is w6.5%. Achieving this threshold  Nitric oxide synthase
should be balanced against the increasing apprecia-  Plasminogen activator inhibitor 1 (PAI-1)
tion that glycemic targets for the prevention of  Peroxisome proliferator-activated receptor (PPAR)
diabetes-related complications need to be individual-  Type 4 collagen
ized for each patient. Tight glycemic control clearly  3-Adrenergic receptor
 Vascular endothelial growth factor (VEGF)
has been shown to reduce the incidence of micro- or  Engulfment and cell motility 1 (ELMO1)
macroalbuminuria. However, it is only in very recent

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Markers of DKD Development and Progression

population with type 2 diabetes, but not type 1 dia- contrast to the meta-analysis mentioned, a single-
betes, and the strongest associations were found center study from the Joslin Diabetes Center found
among Asians.27 no evidence that hyperltration estimated by cystatin
The more contemporary approach to identifying C level predisposes to the development of micro-
genes that are linked to the development of DKD is albuminuria in type 1 diabetes.34 The exact reasons
to perform a genome-wide association study. This for these disparate ndings are not known. However,
involves identifying associations between single- it is possible that a calendar effect may explain the
nucleotide polymorphisms and the DKD phenotype negative ndings in the Joslin Diabetes Center study.
using case-control methodology. Recent studies have That is, improvements in control of glucose levels,
linked specic genetic variants to different DKD BP, and dyslipidemia over the last 20 years may have
phenotypes. For instance, in type 2 diabetes, gene diminished the role of hyperltration as a predictor of
variants associated with a decrease in albuminuria microalbuminuria.
may be different from those associated with an in- Studies of hyperltration in type 2 diabetes are
crease in GFR in advanced DKD.28 For patients with more difcult to interpret than those in type 1 diabetes
type 2 diabetes, reduced GFR has been linked with because of the effects of age and obesity on GFR. A
variants in the engulfment and cell mobility (ELMO1) correction for age-related loss of GFR can be made
gene.29 In type 1 diabetes, decreased GFR has been by subtracting 1 mL/min/1.73 m2 per year after 40
associated with variants in genes controlling matrix years of age. In obese individuals with type 2 dia-
metalloproteinases and with endothelial nitric oxide betes, there also is a continuing debate about whether
(NO). Despite these advances, both candidate gene GFR should be indexed for body surface area. In a
studies and genome-wide linkage studies to date have study involving Pima Indians with type 2 diabetes
failed to identify a major gene that explains the strong with average age younger than 50 years, early GFR
familial clustering of DKD in either type 1 or type 2 loss ($3.3% per year) occurred over 4 years in 88
diabetes. participants and kidney function remained stable in
Epigenetics is a potential mechanism that links 107 participants. Hyperltration at baseline, dened
genes and the environment with DKD.30 Potential as GFR $ 154 mL/min, was present in 53% of par-
epigenetic markers for DKD progression have been ticipants with early GFR loss and a similar proportion
identied by comparing DNA methylation in in- of participants with stable kidney function (48%).
dividuals with or without DKD. Different levels of Therefore, this study does not support the concept that
DNA methylation in individuals with DKD have been hyperltration predisposes to early GFR decline.35
linked with a predisposition to ESRD.31 Of possible However, it should be noted that mean body mass
relevance to DKD, methylation and demethylation of index (BMI) was .30 kg/m2 in both study groups,
the proximal promoter region of the genes, which and if GFR were expressed per body surface area,
regulate ACE expression, have been shown to upre- most of these individuals would not be classied as
gulate or silence ACE expression.32 However, pro- having hyperltration.
spective studies are required to show that epigenetic The risk of the development of micro- or macro-
factors predispose patients to DKD. albuminuria in hypertensive individuals with type 2
diabetes, with or without persistent hyperltration,
EARLY CHANGES IN GFR has been examined over a 4-year follow-up period.
From the 90 individuals with hyperltration at base-
Hyperltration line, 47 still displayed a state of hyperltration after
Hyperltration is a common nding in people with a further 6 months of observation. Of these in-
type 1 diabetes and poor glucose control. There is no dividuals, 11 (23.4%) progressed to the development
universally accepted denition of hyperltration, but of micro- or macroalbuminuria compared with 53 of
a common approach is to use a GFR threshold that is the 502 (10.6%) individuals for whom hyperltration
2 standard deviations above the mean GFR in patients was ameliorated at 6 months or who had normol-
with normal glucose tolerance to signify a state of tration at baseline (HR, 2.16; 95% CI, 1.13-4.14).36
hyperltration. In most individuals, this condition Due to the short study duration, it was not possible
responds to an improvement in glucose control. to determine whether amelioration of hyperltration
However, in a proportion of patients, hyperltration results in slower progression to a GFR threshold of
persists for several years. A recent meta-analysis of 12 60 mL/min/1.73 m2.
studies (6 prospective and 6 retrospective) of patients In summary, to date, there is no evidence that
with type 1 diabetes and hyperltration concluded hyperltration predicts the development of
that hyperltration predisposes to the development of GFR , 60 mL/min/1.73 m2. Further long-term stu-
micro- or macroalbuminuria (HR, 2.3) compared to a dies of GFR gradients therefore are required to
concurrent (normoalbuminuric) control group.33 In establish whether hyperltration ultimately leads to

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MacIsaac, Ekinci, and Jerums

decreased kidney function, after adjustment for gly- long-term study of 195 Pima Indians with type 2
cemic control and other confounders. Although the diabetes, early kidney function decline was dened as
state of hyperltration has been shown to predict the a rate of GFR loss $ 3.3% per year over 4 years. In
progression of albuminuria in many, but not all, this study, GFR was measured directly by urinary
studies, the concept that hyperltration predisposes to clearance of iothalamate and expressed as milliliters
the development of CKD stage 3, that is, GFR decline per minute (no correction for body surface area).
to ,60 mL/min/1.73 m2, remains to be proved.8 The prevalence of early kidney function decline
during this initial period was 32% in participants
Early Kidney Function Decline
with normal urinary ACRs at baseline, 42% in those
For type 1 diabetes, early decline in kidney func- with microalbuminuria, and 74% in those with mac-
tion is dened as a yearly decrement in GFR . 3.3%; roalbuminuria (P , 0.001). Subsequent follow-up
this corresponds to the 97.5 percentile of the decline showed that the cumulative incidence of ESRD 10
in creatinine clearance in nondiabetic normotensive years after the initial period was 41% in those with
whites in the Baltimore Longitudinal Study on Ag- early kidney function decline and 15% in those
ing.37 Using this denition, the rst Joslin Kidney without. It was concluded that an early decline in
Study followed up trends in kidney function over GFR often happens before macroalbuminuria occurs,
4 years in 279 participants with type 1 diabetes with but a decline predictive of ESRD is highly dependent
new-onset microalbuminuria. To compare the annual on the development of macroalbuminuria.35
percentage of change in GFR in this cohort (mean
baseline GFR, 155 mL/min/1.73 m2) with a control ALBUMINURIA
group of normoalbuminuric patients (baseline GFR,
143 mL/min/m2), the authors calculated the reciprocal Prognostic Signicance of Progression and Remission
of serum cystatin C concentration. There was early of Microalbuminuria
kidney function decline in 31% of microalbuminuric Studies dating back to the early 1980s reported that
and 9% of normoalbuminuric patients (P , 0.001).38 microalbuminuria is associated with a 60%-80% risk
The frequency of early kidney function decline was of the development of overt proteinuria over 6-14
related to the trajectory of AERs: a higher frequency years in patients with type 1 diabetes. However, more
of early GFR loss (67.7%) was seen in participants recent studies have raised questions about the prog-
for whom AERs doubled during the study compared nostic signicance of microalbuminuria. The issue was
with those with stable microalbuminuria (32.2%) raised rst following a pooled analysis by Caramori
and participants with halving of microalbuminuria et al40 that demonstrated that the cumulative incidence
(16.2%). The implication of these ndings is that of remission of microalbuminuria varies from 0%-43%
early GFR decline and AER trends are separate pro- in type 1 diabetes, and that rates of progression to
cesses at an early stage, but become more closely macroalbuminuria are only w30%. Other studies
coupled at later stages of DKD. have detected microalbuminuria remission rates of
In a long-term analysis of the DCCT/EDIC 21%-64% in patients with type 1 or type 2 diabetes.5
cohort performed over 15 years in participants with In some, but not all, studies showing substantially
type 1 diabetes, a persistent decline in eGFR to higher rates of microalbuminuria remission, ndings
,60 mL/min/1.73 m2 was observed in 89 of 1,439 have been linked to the use of RAAS blockers. This
participants, of whom 21 had normoalbuminuria at may be explained by the almost universal treatment
baseline. When expressed per 1,000 person-years, of patients with microalbuminuria with these agents,
this translated to 36.1 macro-, 1.3 micro-, and 1.0 the observational design of most of the studies, and
normoalbuminuric participants progressing to CKD insufcient documentation of persistent microalbu-
stage 3.39 The prognostic signicance of early GFR minuria at baseline in some studies. Therefore, in the
loss in type 1 diabetes has been highlighted in a present treatment environment, microalbuminuria is
recent study from the Joslin Diabetes Center involving linked more frequently to remission/regression rather
proteinuric patients. An early accelerated loss of than the development of overt proteinuria.
GFR, which occurred in approximately two-thirds In results from the STENO-2 study, intensive
of patients, was found to be a better predictor of multifactorial intervention was associated with a
ESRD development over a 5-year observational period lower rate of progression from microalbuminuria to
than HbA1c, BP, and ACR values at baseline. In overt nephropathy over 8 years in participants with
contrast, for patients without an early accelerated type 2 diabetes. However, the rate by which isotopi-
decline in GFR, risk for the development of ESRD was cally measured GFR declined was similar whether
considered to be negligible.9 participants received conventional or intensive multi-
The relationship between early renal function loss factorial intervention. A relationship between decline
and AER also has been studied in type 2 diabetes. In a in AER and slower pace of GFR decline was apparent

S46 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 HPLC overestimates AER in the normo- to micro-
results from groups treated by the conventional or albuminuric range. For example, in community
intensive strategies.41 Similarly, a post hoc analysis of studies such as PREVEND (Prevention of Renal and
the IRMA-2 (Irbesartan in Patients With Type 2 Vascular End-Stage Disease) and AusDiab (Austra-
Diabetes and Microalbuminuria) trial has shown that lian Diabetes, Obesity and Lifestyle), similar in-
reductions in albuminuria within the microalbuminuric creases in HPLC-assayable albumin were seen in
range are linked to a slower decline in eGFR. However, participants with normoalbuminuria regardless of
this relationship has been demonstrated only when whether they also had diabetes.48,49 In healthy in-
the albuminuria and GFR relationship was analyzed dividuals, the albumin peak on size-selective HPLC
after combining irbesartan- and placebo-treated in- consists of w70% albumin, with the remaining
dividuals.42 In another observational study of micro- 30% reecting globulins such as transferrin and
albuminuric individuals with type 1 diabetes, an early a1-glycoprotein. Use of HPLC-assayed albumin as a
decrease in GFR happened in 68%, 32%, and 16% of marker of progression would require determining
participants with AER progression, lack of change, or appropriate thresholds based on HPLC-measured
regression, respectively, over the 8- to 12-year obser- albuminuria that would allow the transition from
vation period.38 normoalbuminuria to micro- and macroalbuminuria to
As opposed to the observational studies mentioned be recognized in long-term studies.
in the preceding, a pooled analysis of interventional
studies has demonstrated that the initial decline in Dissociation Between Albuminuria and GFR
AER with antihypertensive therapy is not a predictor In the past decade and a half, worsening AER as a
of the subsequent decrease in GFR during the denitive predictor of diabetic CKD has come under
microalbuminuric phase for patients with type 1 or re. First, spontaneous remission of microalbuminuria
type 2 diabetes. In contrast, AER and GFR responses has been reported in .50% of patients with diabetes,
in patients with type 1 or type 2 diabetes and late as described previously. Second, early GFR decline
nephropathy were found to be closely correlated. now can be detected at the time of incident micro-
These data suggest that the full relationship between albuminuria, substantially before the onset of overt
current therapies, microalbuminuria, and GFR still nephropathy. These studies have been facilitated by
needs to be elucidated.43 On this basis, the value of the use of markers such as cystatin C that accurately
microalbuminuria as a kidney disease end point in identify a decline in GFR even when the resulting
clinical trials has come under question. level is still . 60 mL/min/1.73 m2. In a recent study
Despite this, observational studies have shown a from the Joslin Diabetes Center, 79 patients with type
continuous relationship between increasing albumin- 1 diabetes, new-onset microalbuminuria, and baseline
uria and declining GFR. This relationship becomes eGFR of 104 mL/min/1.73 m2 were followed up over
evident for increasing albuminuria even before the a 12-year period. In that time, 23 of 79 patients
upper limit of normoalbuminuria is reached.44 reached eGFRs , 60 mL/min/1.73 m2 (CKD stage 3).
Furthermore, albuminuria within the microalbumi- Of these, 12 had AERs at the macroalbuminuria stage,
nuric range has been shown to be associated with but the occurrence of macroalbuminuria was con-
increased risk for the development of ESRD temporaneous with, not before, the development of
compared with normoalbuminuria in a meta-analysis advanced CKD. In the other 11 participants, micro-
of 8 studies with an overall relative risk for ESRD albuminuria persisted or reverted to normoalbumi-
of 4.8 (95% CI, 3-7.5). In a similar fashion, a meta- nuria although they reached CKD stages 3-5.50
analysis of 5 studies involving individuals with type The third strike against AER as a prognostic indi-
2 diabetes found that microalbuminuria was associ- cator comes from 2 studies that appeared in the early
ated with a relative risk of 3.6 (95% CI, 1.6-8.4) 1990s, which found that some patients with diabetes
for the development of ESRD compared with develop a low creatinine clearance (CCr) while never
normoalbuminuria.45 progressing beyond normoalbuminuria. The rst
When interpreting these results, it is salient to study, involving kidney biopsies in type 1 diabetes,
consider the limitations of meta-analyses or pooled found changes of classic diabetic glomerulopathy in a
analyses that include reliance on the quality of the small group of female patients with reduced CCr
individual studies included in the analysis, the fact that (,90 mL/min/1.73 m2) and normal AER.51 The sec-
grouped and not individual patient data usually are ond publication, a study of serial kidney function using
analyzed, the heterogeneity of the individual studies, CCr, showed that w30% of diabetic patients (both
and the possible inuence of publication bias.46 type 1 and type 2) have progressive declines in CCr
The best method for measuring albuminuria, without going from normo- to microalbuminuria.52
immunoassay or high-performance liquid chroma- That study involved 40 patients followed up over
tography (HPLC), has become an issue of debate.47 8-14 years who had normal AERs and CCrs at baseline.

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MacIsaac, Ekinci, and Jerums

Three patterns of AER and CCr became evident. In 15 coat effect; measurement of the diurnal rhythm of
patients, AER increased and CCr was stable; in 13, BP, by which patients can be classied into nocturnal
AER increased and CCr decreased; and in 12, AER dippers and nondippers (reduction in BP , 10%);
stayed in the normoalbuminuric range but CCr and recording BP random variability and ambulatory
declined.52 Dissociation between AER and GFR also heart rate. An abnormal circadian variability in BP in
has been reported in type 2 diabetes.53,54 individuals with diabetes has been linked to a number
The normoalbuminuria/low-GFR pattern occurs of kidney disease outcomes. Nondipping of night-
more frequently in elderly women. Furthermore, time BP values predicts the development of micro-
separate sets of risk factors been reported in recent albuminuria in individuals with type 1 diabetes.58
studies for progression to low GFR versus the devel- Individuals with nondipping of night-time BP who
opment of increased AER; this provides additional have type 1 or type 2 diabetes also are at higher risk
evidence that an increase in AER and decrease in GFR of mortality compared with dippers, even after ac-
are complementary rather than necessary manifesta- counting for traditional cardiovascular disease risk
tions of diabetic CKD. In the UKPDS-74, independent factors.59 Recently, higher 24-hour systolic BP and
risk markers for decreased GFR were female sex, pulse pressure values and reduced night/day BP
smaller waist circumference, age, increased insulin variation have been shown to be independent pre-
sensitivity, and previous sensory neuropathy. How- dictors of DKD progression, dened by increases in
ever, male sex, larger waist circumference, plasma AER and/or doubling of serum creatinine level or the
triglyceride level, low-density lipoprotein (LDL) development of end-stage renal failure in individuals
cholesterol level, HbA1c level, increased white blood with type 2 diabetes.60
cell count, history of smoking, and previous retinopathy On the basis of receiver operating characteristic
were independent risk markers for elevated AER.10 curve analysis, one cross-sectional study of 550 par-
ticipants with type 2 diabetes suggested that the
HYPERTENSION ambulatory BP threshold levels that were associated
In individuals with type 1 diabetes, the cause of with overt proteinuria were 125/75, 110/65, and
high BP generally is DKD, and usually BP starts to 120/75 mm Hg for daytime, night-time, and 24-hour
increase only when the patient progresses from micro- periods, respectively.61
to macroalbuminuria. However, hypertension is found
in about one-third of patients with type 2 diabetes at DYSLIPIDEMIA
the time of diagnosis. Thus, for type 2 diabetes, hy- The role of dyslipidemia in the initiation and pro-
pertension most likely is caused by many factors and gression of DKD still is poorly dened. Results of a
may represent a component of metabolic syndrome. number of cross-sectional and prospective studies
Despite the differences in timing and cause of hy- reveal associations between dyslipoproteinemia, in
pertension in type 1 and type 2 diabetes, a number of particular elevated apoB-100containing lipoprotein
studies have shown that high BP hastens the devel- levels, low high-density lipoprotein (HDL) cholesterol
opment of DKD and that reducing BP slows the loss levels, and albuminuria in individuals with diabetes.
of kidney function. In a post hoc analysis from the ADVANCE Study,
Although the evidence from observational studies low baseline HDL cholesterol levels were associated
is consistent with a continuous relationship bet- with a signicantly higher risk for the development of
ween BP levels and renal and cardiovascular events new microalbuminuria and macroalbuminuria. An
in patients with diabetes, very few interventional effect in the same direction was seen for the outcomes
studies have succeeded in attaining BPs below the of doubling of creatinine level and death related to
130/80mm Hg threshold (or the stricter target kidney disease, but it was not statistically signicant.
of ,120/75 mm Hg if proteinuria is present). In both Interestingly, no association was detected between
the ACCORD-BP trial and INVEST (Interna- baseline HDL cholesterol levels and the development
tional Verapamil-Trandolapril Study), which achieved of retinopathy.62 Furthermore, the use of fenobrate
BPs , 130/80 mm Hg, the results suggested that appears to protect against eGFR loss to a greater extent
further lowering BP does not substantially reduce in individuals with low HDL cholesterol and higher
clinical events versus moderate BP control (equiva- triglyceride levels, whereas the retinal benets of
lent to w135/85 mm Hg).55,56 Of note, the American fenobrate appear to be independent of lipid levels. In
Diabetes Association has recently relaxed its general contrast, in the DCCT/EDIC cohort, HDL cholesterol
systolic BP goal from ,130 to ,140 mm Hg.57 concentrations were not associated with albuminuria,
Data from ambulatory BP monitoring may be of but smaller HDL cholesterol particles were associated
value in estimating the risk of clinical outcomes, positively with increasing albuminuria.63
including kidney events in diabetes. Such data include Regression of microalbuminuria has been shown to
estimating true BP levels and detecting the white- be associated independently with low levels of LDL

S48 Am J Kidney Dis. 2014;63(2)(suppl 2):S39-S62


Markers of DKD Development and Progression

cholesterol and triglycerides in studies from the the basis of the new pathologic classication system of
Joslin Diabetes Center and higher LDL cholesterol diabetic nephropathy.71 However, although morpho-
and triglyceride levels were associated with inc- logic changes such as mesangial expansion and
reasing albuminuria in the DCCT/EDIC cohort.63,64 glomerular basement membrane thickening can be
Furthermore, declining GFR (as measured by CCr) linked to markers of DKD progression such as
has been linked with increasing LDL cholesterol increasing albuminuria and declining GFR, dissocia-
plasma levels, smaller LDL cholesterol size, and tion between structure and function has been described.
elevated triglyceride levels in the DCCT/EDIC When persistent microalbuminuria occurs in type 1
mentioned previously. Studies involving the Finne- diabetes, kidney changes detected on biopsy often are
Diane cohort have suggested that higher triglyceride advanced.72 There also is a great deal of heterogeneity
levels are associated with progressive albuminuria, in biopsy ndings from kidneys of individuals with
whereas total cholesterol was the only lipid parameter type 2 diabetes and microalbuminuria.73
associated with progression to ESRD in an adjusted Furthermore, a recent study of proteinuric patients
Cox regression model that included HbA1c level, sex, with type 2 diabetes and biopsy-proven mesangial
smoking, BP, BMI, duration of diabetes, and eGFR.65 expansion showed that glomerular changes were not
Randomized controlled trials have suggested that associated with the outcome of initiation of mainte-
statins may preserve kidney function and that a nance dialysis therapy or doubling of serum creatinine
greater degree of renal preservation is seen for in- level. In contrast, interstitial changes such as brosis,
dividuals treated with higher, compared with lower, tubular atrophy, and interstitial inammation were
doses of statins who also achieved the greatest re- associated independently with these kidney disease
ductions in LDL cholesterol and triglyceride levels.66 end points.74
In CARDS (Atorvastatin Study for the Prevention of In summary, although some morphologic changes
Coronary Artery Disease Endpoints in Non-Insulin detected on kidney biopsy are linked to functional
Dependent Diabetes Mellitus), treatment with ator- changes, the DKD pathologic ndings that predict
vastatin was found not to be associated with pro- kidney disease outcomes still remain to be clearly
gression or regression of albuminuria, but slowed the dened. Therefore, the use of biopsy ndings as a
annual change in eGFR (w0.2 mL/min/1.73 m2 per marker of disease progression is still not recognized
year; P 5 0.01) compared with placebo.67 as a part of routine clinical practice for the manage-
ment of DKD.
STRUCTURAL MARKERS
Structural and Functional Indexes of Vascular
Kidney Size Dysfunction
Diabetic hypertrophy has been linked to the Patients with type 2 diabetes and eGFR , 60 mL/
development of microalbuminuria and increased risk min/1.73 m2 have higher measures of intrarenal
of developing ESRD when compared with normal- vascular resistance, as measured by Doppler ultra-
sized kidneys.68,69 Kidney hypertrophy most likely sound techniques, compared with patients with
represents a maladaptive response to hyperglycemia eGFR $ 60 mL/min/1.73 m2. However, renal vascular
and the release of various cytokines and growth fac- resistance index is elevated to a similar extent in pa-
tors, such as insulin-like growth factor 1 (IGF-1), tients with type 2 diabetes and eGFR , 60 mL/min/
TGFb, vascular endothelial growth factor (VEGF), 1.73 m2, regardless of albuminuric status (Fig 3).75
and possibly through adenosine monophosphate Higher, compared to lower, baseline indexes of intra-
(AMP)-activated protein kinase, a key player in renal vascular resistance also are associated with a
regulating kidney metabolism that predisposes to the greater subsequent decline in GFR and likelihood for
development of DKD.70 the development of proteinuria in type 2 diabetes.76
Extrarenal vascular dysfunction, as measured by
Kidney Biopsy Findings abnormal echocardiogram ndings and mainly related
The characteristic morphologic changes that are to the nding of diastolic dysfunction, has been
seen in type 1 DKD are glomerular basement mem- associated with reduced kidney function as measured
brane thickening, mesangial expansion, and hyali- by CCr in individuals with type 2 diabetes.77
nosis of the afferent and efferent arterioles. Pathologic Increased aortic stiffness, as assessed by pulse wave
changes in glomerular epithelial cells that are asso- velocity, also has been linked to the development of
ciated with decreased number of podocytes and/or more advanced stages of albuminuria and annual
podocyte foot-process broadening and effacement change in GFR.78 Further evidence of extrarenal
also are seen. The major glomerular feature that pre- vascular disease being linked to reduced kidney
dicts DKD progression is mesangial expansion. It function in diabetes has been provided by a study
therefore is the central morphologic feature that forms using magnetic resonance imaging to detect silent

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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
Figure 3. Intrarenal arterial resistance index in 325 patients risk markers. In addition, endothelial dysfunction z
with type 2 diabetes stratified according to eGFR (ie, ,60 score was found to enhance risk assessment for the
or $60 mL/min/1.73 m2) and albuminuric status (ie, normoalbu- development of macroalbuminuria when combined
minuria [AER , 20], microalbuminuria [20-200], or macroalbumi-
nuria [.200]). Open bars: eGFR , 60 mL/min/1.73 m2 (n 5 93); with albuminuria and whether participants were treated
closed bars: eGFR $ 60 mL/min/1.73 m2 (n 5 232). Repro- with an RAAS blocker.81
duced with permission of the American Diabetes Association
from MacIsaac et al.75 ADVANCED GLYCATION
The accumulation of abnormal indicators of pro-
cerebral infraction, a marker of small-vessel disease. tein glycation is thought to play a key role in the
In that study, multivariate analysis showed that the initiation and progression of DKD. Immunohisto-
composite outcome of a doubling of serum creatinine chemical studies in individuals with DKD have
level or commencing dialysis therapy was signi- shown that AGEs accumulate in the mesangium and
cantly greater for individuals with evidence of silent glomerular capillary wall. The kidney is an important
cerebral infraction compared with those without, regulator of AGE metabolism, and circulating low-
during a follow-up of 7.5 years. Interestingly, the molecular-weight (LMW) AGE levels are elevated
presence of silent cerebral infraction did not increase in patients with diabetes and reduced GFR.82 How-
the risk for the progression of albuminuria.79 ever, the exact role of circulating LMW AGEs in the
Endothelial dysfunction usually is characterized by pathogenesis of DKD is uncertain. More recently, it
an imbalance between vasodilating and vasocon- was shown that serum from patients with type 2 dia-
stricting substances, either produced by or acting betes and various degrees of reduced kidney function
on the endothelium, but also related to perturba- contains high-molecular-weight ligands (.50 kDa),
tions of other endothelial functions, including those mainly protein-bound N-carboxymethyllysine, that
related to coagulation, platelet adhesion, and immune bind and activate the receptor for AGE (RAGE).83
function. A quantiable feature of endothelial dys- Various pathways downstream of RAGE have been
function is the inability of arteries to dilate fully in implicated in the development of diabetic complications.
response to appropriate stimuli. NO, generated by In particular, experimental models of diabetes that over-
the endothelium, is a key substance that regulates express RAGE show evidence of damage to kidney
vasodilation, with asymmetrical dimethylarginine function and structure.84 Unfortunately, the relationship
(ADMA) being the major endogenous inhibitor of between serum AGE concentrations and markers of
NO production.80 reduced kidney function in diabetes still remains to be
One recent study has suggested that ADMA may clearly dened. Despite this, one study has shown that
be a novel and important predictor of DKD progres- levels of protein glycation, oxidation and nitration ad-
sion. In that study, 225 participants with type 2 dia- ducts (measured in plasma ultraltrates with a 12-kDa
betes and with the majority having normoalbuminuria cutoff, and determined by liquid chromatography
and eGFR . 60 mL/min/1.73 m2 had baseline levels tandem mass spectrometry [LC-MS/MS]) are related to
of ADMA measured and then were followed up for 5 albuminuria, but not GFR. In that study, adduct levels
years. Those with higher, compared with lower, were not signicantly different among plasma samples
baseline ADMA levels had a greater chance of pro- collected from individuals with type 1 diabetes and
gressing to a more advanced stage of albuminuria and microalbuminuria who had stable or an early decline in
also had a greater rate of decline in eGFR during GFR decline. However, 3 protein damage adduct residues
follow-up. Furthermore, the relationship between were decreased and 3 were increased in microalbumi-
baseline ADMA levels and increases in albuminuria nuric compared with normoalbuminuric individuals. The

S50 Am J Kidney Dis. 2014;63(2)(suppl 2):S39-S62


Markers of DKD Development and Progression

largest differences were of N-formylkynurenine protein only levels of soluble forms of receptors for tumor
adduct residue and Nv-carboxymethylarginine (CMA) necrosis factor (TNF), for both receptor 1 and re-
free adduct, for which levels were substantially lower in ceptor 2, and Fas (which binds the Fas ligand and
microalbuminuric compared with normaoalbuminuric results in an apoptotic response), were found to be
individuals (P , 0.001 for both).85 associated with GFR in a multivariate analysis.
The potential value of urinary excretion of proteins Interestingly, variation in TNF receptor concentra-
modied by AGEs as biomarkers for albuminuria has tions turned out to be linked more strongly to GFR
been highlighted in recent studies. In participants with than clinical covariates such as age and albumin
both type 1 and type 2 diabetes, it has been shown excretion.88
that that there is a graded relationship between level Subsequent studies have demonstrated the prog-
of albuminuria and urinary AGE-modied proteins that nostic signicance of baseline TNF receptor levels in
is independent of GFR (Fig 4). In particular, urinary individuals with type 1 and type 2 diabetes. In a study
N-carboxymethyllysine excretion was found to be involving participants with type 2 diabetes, baseline
increased in both micro- and macroalbuminuric partic- levels of TNF receptors, especially for the type 1 re-
ipants with type 1 diabetes compared with normoalbu- ceptor, independently predicted progression to ESRD
minuric participants.86 However, it remains to be shown over 12 years. This association was stronger in par-
that changes in urinary protein-bound AGE concentra- ticipants without proteinuria than in those with pro-
tions precede an increase in urinary albumin excretion teinuria and was independent of other markers of
or whether their measurement adds to the prognostic the TNF pathway, markers of endothelial dysfunction,
ability of traditional DKD progression markers. and markers of systemic inammation, such as IL-6
and C-reactive protein (CRP). It was suggested by
MARKERS OF INFLAMMATION AND
the authors of that study that high circulating TNF
OXIDATIVE STRESS receptor levels could be a new predictor of ESRD
Recent studies from the Joslin Diabetes Center have that has a more powerful predictive ability than
examined the contribution of inammatory processes proteinuria.89
in the progression of kidney damage in people with A further study demonstrating the potential value
diabetes. In one study, 5 inammatory markers, of measuring circulating TNF receptor has been per-
interleukin 6 (IL-6), IL-8, monocyte chemoattractant formed in participants with type 1 diabetes with
protein 1 (MCP-1), interferon ginducible protein (IP- normo- or microalbuminuria and without evidence of
10), and macrophage inammatory protein 1d, were hypoltration at baseline. The cumulative incidence
measured in urine samples collected from individuals for reaching stage 3 CKD for individuals with the
with type 1 diabetes. Baseline urinary concentrations highest TNF type 2 receptor quartile was found to be
of these inammatory markers were found to be 60% after 12 years compared with only 5%-19% for
signicantly higher in those with an early progressive the other quartiles. In Cox proportional hazards
kidney function decline (.3 mL/min/1.73 m2 per year) analysis, participants with TNF type 2 receptor values
compared with those who displayed stable kidney in the highest quartile were 3-fold more likely to
function.87 By multivariate analysis, those with more experience an early decline in kidney function than
than 2 elevated marker levels were found to be more individuals in the other quartiles (HR, 3.0; 95% CI,
than 5 times as likely to have an early progressive 1.7-5.5). In this study, risk of progression to stage 3
decline in kidney function. CKD associated with high TNF type 1 receptor levels
Another study investigated the relationship of 12 was slightly less than that associated with high TNF
serum markers of inammation and apoptosis to GFR type 2 receptor levels.90
estimated from cystatin C level in a cross-sectional A recent report based on the DCCT/EDIC also has
analysis of individuals with type 1 diabetes. However, suggested that high levels of inammatory markers,

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

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MacIsaac, Ekinci, and Jerums

especially E-selectin and circulating TNF receptors 1 Clinical studies evaluating the role of TGFb have
and 2, are important independent predictors of the found an inconsistent link between plasma levels of
development of macroalbuminuria.91 this cytokine and indexes of kidney health in people
In summary, these studies suggest that serum TNF with diabetes, which may be related to platelet
receptor levels are linked to the development and degranulation. In contrast, urinary TGFb levels
progression of DKD. Circulating TNF receptor levels consistently have been shown to be elevated in in-
may even be a stronger predictor of declining GFR dividuals with DKD compared with those without.95
than albuminuria. However, the exact mechanisms Moreover, recent studies also have reported that uri-
linking TNF receptors with DKD and whether nary TGFb excretion is attenuated by ACE inhibition
modulating their levels improves the prognosis of or cholecalciferol in type 2 diabetic patients with
patients with DKD remain unknown. DKD.96
Experimental studies have highlighted the impor- Urinary CTGF level has been found to closely
tance of oxidative stress, in particular the generation correlate with ACR. In this analysis, both parameters
of ROS, derived largely from hyperglycemia- were expressed on a logarithmic scale; urinary CTGF
triggered overactivation of the NADPH (reduced increased 10-fold in microalbuminuric versus nor-
nicotinamide adenine dinucleotide phosphate) oxidase moalbuminuric patients and 100-fold in macro-
(NOX) family of free radicalproducing enzymes albuminuric versus normoalbuminuric patients. A
in DKD.92 Mitochondrial ROS production has been positive relationship between urinary TGFb and
identied as the key regulator that links the 4 CTGF levels also was evident.97
major pathways implicated in the development of Larger studies have provided more evidence that
hyperglycemia-driven tissue damage, in other words, urinary excretion of CTGF is related to albuminuria
the polyol, hexosamine, PKC, and AGE pathways. and also to GFR in type 1 diabetic patients with
However, clinical studies have not clearly demon- diabetic nephropathy.98 In particular, one study has
strated that markers of oxidative stress, although shown that baseline plasma CTGF levels are associ-
elevated in individuals with diabetes compared with ated independently with risks of ESRD and mortality
those without diabetes, such as urinary 8-hydroxy-20 - in individuals with type 1 diabetes. However, this
deoxyguanosine (8-OHdG) levels, offer additional relationship was evident in only macroalbuminuric
prognostic information in relation to the development individuals compared with normalbuminuric in-
or progression of DKD over and above established dividuals. Plasma CTGF levels also were found to
risk markers. In one study, higher, compared with signicantly improve the prediction of ESRD and
lower, urinary 8-OHdG levels were associated with mortality for macroalbuminuric individuals in addi-
progression of albuminuria in participants with type 2 tion to conventional risk factors.99
diabetes. Furthermore, multivariate logistic regres- It recently has been shown that genetic variation
sion analysis suggested that urinary 8-OHdG levels in the CTGF-945 polymorphism, which has been
were the strongest predictor of progression of albu- linked previously to susceptibility to systemic scle-
minuria among several established risk factors.93 rosis, is not associated with the presence of DKD or
However, another more recent study has suggested abnormal echocardiogram ndings after adjustment
that measuring urinary 8-OHdG did not improve the for multiple covariates in individuals with type 2
ability to determine which patients are at risk of pro- diabetes (Fig 5).100 These ndings have been con-
gressive DKD over and above measuring urinary rmed in another study, which interestingly showed
ACR.94 that CTGF-945 polymorphism was not associated
NF-E2-related factor (Nrf2) is the primary tran- with plasma CTGF level.101 Thus, further studies are
scription factor that regulates intracellular antioxi- required to assess the roles of urinary CTGF and
dants and possibly its modulation will prove to TGFb as markers of progression of DKD.
have a renal protective effect. Unfortunately, although
experimental and some clinical studies suggested that SERUM CYSTATIN C
the Nrf2 modulator bardoxolone may have DKD An alternative approach to current methods of esti-
protective effects, a large clinical study involving mating GFR is to use a different analyte to creatinine
bardoxolone recently was ceased due to adverse for developing GFR predicting equations. More than
outcomes in participants randomly assigned to receive 25 years ago, Grubb et al102 proposed that cystatin C
bardoxolone. could be used as a potential marker of GFR based on
the fact that, unlike creatinine level, cystatin C levels
CYTOKINES generally are not inuenced by extrarenal factors such
Studies of experimental diabetes have hinted that as age, sex, diet, and muscle mass. Cystatin C is an
probrotic cytokines, such as CTGF and TGFb, may LMW (13.3-kDa) protein that is produced at a constant
be markers of progression of DKD. rate by all nucleated cells, freely ltered by the

S52 Am J Kidney Dis. 2014;63(2)(suppl 2):S39-S62


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 creatinine-based eGFR had a lower risk than those
proximal tubule. However, it is only recently that with concordant staging (HR, 0.30; 95% CI, 0.13-
studies have supported the measurement of serum 0.68).106
cystatin C as a simple, reliable, and accurate marker of In another study,107 serum cystatin C levels were
GFR in individuals with diabetes.103 In particular, es- found to be a better predictor of ESRD than directly
timations of GFR based on cystatin C level have been measured GFR (iothalamate clearance) or serum
shown to more accurately reect changes in meaured creatinine level. In that study, 234 Pima Indians were
GFR within the normal-to-high range than creatinine- followed up for a median of 10.7 years, with 68
based estimates of GFR.104,105 (29%) developing ESRD. In analyses adjusted for
Two recent studies also have shown that mea- age, sex, diabetes duration, height, weight, HbA1c
surement of serum cystatin C signicantly improves level, and ACR, the reciprocal of serum cystatin C had
the ability to predict which patients with diabetes are the highest area under the receiver operating charac-
at the greatest risk of progression to ESRD compared teristic curve (AUROC) of 0.845 6 0.026. Models
with existing methods for estimating kidney function. with measured GFR or the reciprocal of serum creati-
In one study,106 more than 1,000 patients from 3 nine had similar AUROCs, which were lower than the
separate cohorts with both type 1 and type 2 diabetes model with the reciprocal of cystatin C alone (P 5 0.02
and CKD stages 1-3, based on the CKD-EPI (CKD and P 5 0.03, respectively). It still is unclear whether
Epidemiology Collaboration) creatinine equation, the superior predictive value of serum cystatin C levels
were followed up for 8-10 years for the onset of for ESRD in patients with diabetes is due to an
ESRD, which occurred in 364 participants. CKD enhanced ability to reect true GFR beyond directly
staging also was performed using an eGFR formula measured GFR or its abilty to incorporate nonrenal
based on cystatin C level and was found to signi- inuences on progressive kidney failure.107
cantly improve ESRD risk statication. Concurrent In summary, recent work suggests that measure-
CKD staging was seen in 62% of participants. How- ment of serum cystatin C provides a simple and ac-
ever, participants given a higher (more advanced) curate method for detecting early decline in kidney
CKD stage by cystatin C versus creatinine-based function and subsequent trends in kidney function for
eGFR had a signicantly higher risk of ESRD than individuals with diabetes. Evidence also is starting to
those with concordant staging in all 3 cohorts (HR, emerge to suggest that in comparision to creatinine,
2.3; 95% CI, 1.8-3.1). Similarly, patients at a lower measurement of cystatin C can strengthen the asso-
(less advanced) CKD stage by cystatin C versus ciation between eGFR and risk of death and ESRD.

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MacIsaac, Ekinci, and Jerums

Although extrarenal factors may inuence cystatin L-FABP levels were found to be a strong and inde-
C levels, the inuence of these factors on cystatin pendent predictor of progressive DKD.111 In a cross-
C levels is likely to be much smaller than their cor- sectional study of participants with type 2 diabetes,
responding effects on creatinine levels. However, heart fatty acid-binding protein (H-FABP), a marker
although cystatin C is emerging as a very promising of distal tubular damage, was the only tubular marker
alternative to creatinine-based methods for estimating associated with eGFR after adjustment for other risk
GFR, further studies are needed to investigate the markers of progressive DKD.112
inuence of nonrenal factors on cystatin C levels. An inconsistent relationship between urinary
Previous problems with cystatin C assays, including a tubular markers and regression or progression of
drift in calibration of the measurement procedures and albuminuria has been reported. One study involving
lack of standardization against against an international participants with type 1 diabetes showed that regres-
reference, have recently been addressed, which sion of microalbuminuria was associated with lower,
should help to clarify the role of cystatin C as a compared to higher, urinary levels of both KIM-1 and
marker of GFR. It also is necessary to develop NGAL, with this relationship being independent of
reference ranges for cystatin C levels in large numbers other clinical or biochemical variables. In contrast,
of individuals with diabetes across a wide range of another study involving participants with type 2 dia-
kidney function. Given these limitations, together betes did not nd a relationship between levels of
with the added expense of measuring cystatin C tubular biomarkers and progression from normo- to
compared to creatinine and the lack of access to microalbuminuria or regression of micro- to normoal-
laboratories that routinely measure cystatin C, buminuria.110 As mentioned, increased urinary levels
creatinine-based methods will almost cetainly remain of L-FABP have been linked to progressive albumin-
the most clinically applicable way of estimating GFR uria. One study has shown that urinary L-FABP levels
in the immediate future. independently predict the development of micro-
albumuria in individuals with type 1 diabetes.113
TUBULAR MARKERS As mentioned in the previous section, cystatin C
During the last 10 years, it has been shown that usually is freely ltered by the glomerulus and then
tubulointerstitial, in addition to glomerular, damage is completely reabsorbed and metabolized by the prox-
an important factor in the progression of DKD.108 The imal tubule. Therefore, the appearance of cystatin in
urinary proximal tubular markers that have been urine increasingly is becoming recognized as a marker
studied most vigorously include kidney injury mole- of tubular dysfunction. The urinary cystatin C to
cule 1 (KIM-1), neutrophil gelatinase associated lip- creatinine ratio has been shown to independently
ocalin (NGAL), and liver-type fatty acid-binding predict the development of CKD stage 3 in in-
protein (L-FABP). dividuals with type 2 diabetes after adjustment
One study has shown that higher baseline levels of for a number of covariates. However, in a separate
KIM-1 and NGAL, but not L-FABP, are associated analysis of individuals who initially had eGFRs
with a faster decline in GFR in proteinuric patients . 60 mL/min/1.73 m2 and normoalbuminuria, uri-
with type 1 diabetes. However, after accounting for nary cystatin C to creatinine ratio was not associated
known progression promoters of DKD such as albu- independently with the rate of decline in eGFR.114
minuria, diabetes duration, glycemic control, and BP, In summary, although higher levels of tubular injury
this relationship was found to lose signicance.109 markers may be associated with a faster decline in GFR
Similarly, in a 4-year follow-up study of participants and progression or remission of albuminuria in some
with type 2 diabetes but with the majority having studies, it has been difcult to show that measurement
eGFRs $ 60 mL/min/1.73 m2 and normoalbumi- of tubular markers offers additional prognostic infor-
nuria, higher urinary baseline levels of the tubular mation over and above established risk markers.
biomarkers KIM-1 and glycoprotein nonmetastatic Members of the FABP family are emerging as the
melanoma B (Gpnmb) were found to be associated tubular markers with the greatest chance of offering
with a faster decline in eGFR in univariate analysis. added predictive value for progressive DKD over and
However, this association was no longer signicant above that offered by established risk markers.
after adjusting for known risk factors, such as baseline
albuminuria, BP, eGFR, or HbA1c level.110 URIC ACID
Despite this, urinary L-FABP levels have been During the last 5 years, evidence has accumulated
shown to be associated with progression of DKD, to suggest that increased levels of serum uric acid are
dened as progressive albuminuria or the develop- an independent risk factor for progressive DKD.
ment of ESRD. For individuals with type 2 diabetes Interpreting studies that have attempted to link
who were followed up for 4 years and who had an elevated serum uric acid levels with DKD has been
initial eGFR . 60 mL/min/1.73 m2, high urinary complicated by the confounding inuences of GFR

S54 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. role. To date, no clinical study has clearly shown that
Despite this, many observational studies have shown serum uric acid levels start to increase before GFR
a statistically independent association between uric declines in individuals with diabetes who are destined
acid level and reduced GFR or the development of to develop DKD. This area of research also awaits
proteinuria. results of clinical studies examining the impact of
In a cross-sectional study of participants with type lowering serum uric acid levels to indexes of kidney
1 diabetes who had normo- or microalbuminuria, uric health in people with diabetes.121
acid levels in the high-normal range were found to be
associated independently with reduced GFR as PROTEOMICS
measured by cystatin C level.115 In another study, the Recently, techniques of urinary proteome analysis,
same group of researchers were able to demonstrate which have taken a systematic approach to the iden-
an independent dose relationship between baseline tication and quantication of urinary proteins, have
uric acid levels and risk of early GFR loss and risk of been used in an attempt to identify novel urinary
developing CKD stage 3 in participants with type 1 markers for the development and progression of
diabetes who were followed up for 4-6 years. Inter- DKD. In particular, the prole of the LMW protein
estingly, uric acid levels were not associated with risk fraction (,2,000 Da) in urine, which is composed of
of progression or regression of albuminuria in that intact LMW proteins and peptide fragments, has been
study.116 examined.
Studies demonstrating a relationship between A sophisticated analysis of the urinary peptidome
elevated uric acid levels and the development of suggested that it may be possible to identify which
microalbuminuria have remained elusive. In a study patients with microalbuminuria and type 1 diabetes
from the Steno Diabetes Centre, baseline uric acid are at greatest risk of experiencing an early decline in
levels were predictive of the development of macro- but kidney function. Of the peptides identied by liquid
not microalbuminuria in an inception cohort study of chromatography matrix-assisted laser desorption/
263 participants with type 1 diabetes followed up for ionization time-of-ight mass spectrometry, 3 were
18 years.117 In that study, the HR for the development present at lower levels in urine of patients with early
of macroalbuminuria was 2.37 (95% CI, 1.04-5.37) for kidney function decline (fragments of a-1(IV)
every 100-mmol/L increase in serum uric acid level collagen, a-1(V) collagens, and tenascin-X) and 3
(P 5 0.04). In another study involving participants were increased (fragments of inositol pentaki-
with type 1 diabetes, baseline uric acid levels were sphosphate 2-kinase, zona occludens 3, and FAT
associated with the development of albuminuria at a tumor suppressor 2) compared with individuals with
6-year follow-up visit in an analysis that examined stable kidney function.122
the risk of developing micro- or macroalbuminuria.118 A large cross-sectional study has identied pepti-
In multivariate analysis, each 60-mmol/L increase in dome patterns that have differentiated patients with or
serum uric acid level at baseline was associated with without diabetes, even in the absence of DKD, using
an 80% increased risk for developing albuminuria. capillary electrophoresis coupled with mass spec-
For individuals with type 2 diabetes, normal kidney trometry. Furthermore, urinary biomarkers also could
function, and without overt proteinuria, baseline uric distinguish individuals with type 1 diabetes from
acid levels were associated with the development of those with type 2 diabetes. Specic collagen frag-
CKD (dened as overt proteinuria or eGFR , 60 mL/ ments were associated with diabetes and type of
min/1.73 m2) over a 5-year follow-up. After adjusting diabetes, suggesting that collagen turnover and
for multiple covariates, each 1standard deviation extracellular matrix changes are indicative of the
increase in uric acid levels was associated with a 21% molecular pathophysiology of diabetes. The most
increased risk of CKD.119 Furthermore, post hoc pronounced diabetes-associated urinary proteome
analysis of the RENAAL trial has demonstrated that changes were a signicant reduction in specic
the degree of serum uric acid reduction at 6 months collagen a-1 (I) and (III) fragments, with these
posttreatment with losartan is linked to a lower risk of changes being signicantly dramatic in individuals
doubling of serum creatinine level or the development with type 2 diabetes than in those with type 1 dia-
of ESRD in proteinuric individuals with type 2 dia- betes. Additional biomarkers that were identied
betes.120 This relationship also was shown to be in- included those implicated in inammatory and pro-
dependent of other risk markers, such as eGFR and thrombotic processes.123
albuminuria. Using this methodology, 2 further studies have
Although the exact mechanisms linking elevated investigated the prognostic signicance of the so-
uric acid levels with DKD are not fully understood, called CKD biomarker classier (CKD273) to iden-
contributions from inammatory pathways, endothe- tify patients at risk of progressive DKD. This urinary
lial dysfunction, and hypertension most likely play a peptide classier, consisting of 273 dened urinary

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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,
Figure 6. Time course of the CKD273 classifier and albumin attributed largely to collagen fragments, or whether
excretion rate (AER) in individuals with type 1 or type 2 diabetes novel biomarkers, identied by capillary electropho-
who either progressed (grey line) or did not progress (black line) resis coupled with mass spectrometry technology, will
to develop macroalbuminuria. The dotted line depicts the cutoff
of 0.343 arbitrary units for the CKD273 classifier, the score be the most appropriate path toward earlier diagnosis
that previously has been shown to discriminate between of DKD.
CKD273 casepositive or negative individuals, and 20 mg/min
for AER, the accepted threshold for the development of microal- OTHER NOVEL MARKERS
buminuria. Reproduced with permission of the American Dia-
betes Association from Zurbig et al.125 Fibroblast growth factor 23 (FGF-23) has been
implicated as a risk marker for progressive CKD.
peptides, previously has been shown to be a good Serum FGF-23 levels have been shown to be an in-
classier of individuals with and without CKD dependent predictor of an outcome comprising death,
regardless of cause.124 In a longitudinal study of 16 doubling of serum creatinine level, and/or dialysis in a
participants with type 1 or 2 diabetes who all devel- small study of 55 participants with type 2 diabetes and
oped macroalbuminuria, the transition from normo- to macroalbuminuria who were followed up for 30
microalbuminuria occurred 3.4 6 2.1 years before months. FGF-23 is emerging as an important regu-
progression from micro- to macroalbuminuria. In lator of phosphate metabolism, but interestingly, in
contrast, a CKD273 classier-positive pattern was the mentioned study, FGF-23 levels were not asso-
detected at 4.9 6 2.2 years (P 5 0.016) before the ciated with levels of markers of bone metabolism or
transition from micro- to macroalbuminuria (Fig 6). phosphate.127
Discrimination between a cutoff score for being DKD usually is associated with the accumulation
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- IV collagen. One study has shown that high urinary
opment of microalbuminuria in participants who were excretion of type IV collagen is associated indepen-
destined to develop macroalbuminuria. This suggests dently with an annual decline in eGFR over an 8-year
that patients with normoalbuminuria and a CKD273 observational period.128 A relationship between uri-
classierpositive pattern are at increased risk for the nary type IV collagen and eGFR decline also was
development of macroalbuminria (overt diabetic ne- seen in normoalbuminuric individuals. Measurement
phropathy) and can be identied as such before the of urinary type IV collagen therefore may prove to be
development of microalbuminuria. In urine of patients useful in identifying people at risk of a progressive
with both type 1 and type 2 diabetes, fragments of decline in GFR in the absence of increasing albu-
collagen were detected in lower levels for those who minuria. How these ndings of increased full-length
developed macroalbuminuria compared with those collagen urinary excretion relate to reports of
who did not.125 reduced collagen fragments, detected in proteomic
Furthermore, CKD273 classier score has been studies, awaits further investigation.
shown to be associated independently with progres- Quantifying podocyte injury through the measure-
sion of albuminuria in a case-control study involving ment of urinary markers has been proposed as a novel
participants with type 2 diabetes. In that study, cases way of identifying people at risk of progressive DKD.

S56 Am J Kidney Dis. 2014;63(2)(suppl 2):S39-S62


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 High variability, low specificity for DKD; spontaneous
range predict ESRD regression and DAER within the microalbuminuric range
s DGFR
GFR Best measure of kidney function Routine methods for accurately estimating GFR in the
normal-high range are still lacking
Glucose An important marker because hyperglycemia is the Targets still to be optimally defined; evidence documenting
initiator of DKD that intensive glycemic control prevents ESRD is sparse
Blood pressure An important promoter of DKD; also important in CV Targets are still to be optimally defined
risk reduction
Lipids Important in CV risk reduction; lipid-modifying The relationship between components of the lipid profile
agents may have renal-protective effects and risk for DKD progression is not optimally defined
independent of changes in lipid profile
Soluble TNF Circulating levels of TNF receptors have been The relationship between TNF receptors and ESRD
receptors shown to predict ESRD and possibly have a more remains to be confirmed in various patient populations
powerful predictive ability than proteinuria attending different centers
Uric acid Easy to measure; levels also may relate to CV risk Kidney disease outcome intervention studies to target uric
acid levels are still required
Tubular markers Easy to measure in urine sample The prognostic significance of their measurement over
and above established risk factors remains to be fully
defined
Urinary proteome The appearance of the CKD273 biomarker Clinical assays are lacking
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 Expensive and the standardization of assays is not yet
methods and possibly directly measured GFR universal
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.

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

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


MacIsaac, Ekinci, and Jerums

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type 2 diabetes (UKPDS 35): prospective observational study.
Support: The development of this journal supplement was
funded by Novo Nordisk. The authors work described in this BMJ. 2000;321(7258):405-412.
manuscript was supported by a Regional Diabetes Support grant 15. The Diabetes Control and Complications Trial Research
from Novo Nordisk and the St Vincents Hospital, Melbourne Group. The effect of intensive treatment of diabetes on the
Research Endowment Fund. No other honoraria or remuneration development and progression of long-term complications in
were received for this work. Technical editing was provided by insulin-dependent diabetes mellitus. The Diabetes Control and
Watermeadow Medical, funded by Novo Nordisk. Costs associ- Complications Trial Research Group. N Engl J Med.
ated with publication were funded by Novo Nordisk. 1993;329(14):977-986.
Financial Disclosure: Prof MacIsaac has received honoraria 16. Zoungas S, Chalmers J, Ninomiya T, et al. Association of
for lectures and travel support from Eli Lilly, Novo Nordisk, HbA1c levels with vascular complications and death in patients
Sano Aventis, Astra Zeneca, Merck Sharp & Dohme, Servier, with type 2 diabetes: evidence of glycaemic thresholds. Dia-
Boehringer Ingleheim, and Novartis and has received research betologia. 2012;55(3):636-643.
grants from Novo Nordisk. Dr Ekinci has received honoraria for 17. DCCT EDIC Research Group; de Boer IH, Sun W,
lectures from Eli Lilly, Novo Nordisk, and Astra Zeneca. Prof Cleary PA, et al. Intensive diabetes therapy and glomerular ltration
Jerums declares that he has no relevant nancial interests. rate in type 1 diabetes. N Engl J Med. 2011;365(25):2366-2376.
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