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European Journal of Clinical Investigation (2004) 34, 785–796

Review
Blackwell Publishing, Ltd.

New insights into the pathophysiology of diabetic


nephropathy: from haemodynamics to molecular pathology

G. Wolf
University of Hamburg, Hamburg, Germany

Abstract Although debated for many years whether haemodynamic or structural changes are more
important in the development of diabetic nephropathy, it is now clear that these processes
are interwoven and present two sides of one coin. On a molecular level, hyperglycaemia and
proteins altered by high blood glucose such as Amadori products and advanced glycation
end-products (AGEs) are key players in the development of diabetic nephropathy. Recent
evidence suggests that an increase in reactive oxygen species (ROS) formation induced by
high glucose-mediated activation of the mitochondrial electron-transport chain is an early
event in the development of diabetic complications. A variety of growth factors and cytokines
are then induced through complex signal transduction pathways involving protein kinase C,
mitogen-activated protein kinases, and the transcription factor NF-κB. High glucose, AGEs,
and ROS act in concert to induce growth factors and cytokines. Particularly, TGF-β is
important in the development of renal hypertrophy and accumulation of extracellular matrix
components. Activation of the renin-angiotensin system by high glucose, mechanical stress,
and proteinuria with an increase in local formation of angiotensin II (ANG II) causes many
of the pathophysiological changes associated with diabetic nephropathy. In fact, it has been
shown that angiotensin II is involved in almost every pathophysiological process implicated
in the development of diabetic nephropathy (haemodynamic changes, hypertrophy, extra-
cellular matrix accumulation, growth factor/cytokine induction, ROS formation, podocyte
damage, proteinuria, interstitial inflammation). Consequently, blocking these deleterious
effects of ANG II is an essential part of every therapeutic regiment to prevent and treat
diabetic nephropathy. Recent evidence suggests that regression of diabetic nephropathy could
be achieved under certain circumstances.
Keywords Angiotensin II, diabetic nephropathy, growth factors, progression, reactive
oxygen species.
Eur J Clin Invest 2004; 34 (12): 785 –796

Introduction diabetes mellitus type 2, considered 20 years ago as a some-


what benign condition invariably associated with the ‘normal’
Diabetic nephropathy is one of the major causes of end- ageing process, is now the most common single cause of
stage renal disease in the Western world. Although the inci- renal insufficiency in the USA, Japan and Europe [1–3].
dence of nephropathy owing to type 1 diabetes is declining, There are more than 10 million people with diabetes alone
in the USA and it has been estimated that this number will
Department of Medicine, Division of Nephrology, Osteology, double by 2030 [4]. Patients with type 2 diabetes undergoing
and Rheumatology, University of Hamburg, Hamburg, Germany maintenance dialysis require significantly higher financial
(G. Wolf ). resources than those suffering from nondiabetic end-stage
Correspondence to: Gunter Wolf, MD, University of Hamburg, renal diseases. Furthermore, this group of patients has a very
University Hospital Eppendorf, Department of Medicine, poor prognosis on maintenance dialysis owing to extremely
Division of Nephrology, Osteology, and Rheumatology, high mortality caused by cardiovascular events [2,3]. To
Pavilion N26, Martinistraße 52, D-20246 Hamburg, Germany. develop innovative therapeutic concepts to prevent the
Tel.: +49 40/42803–5011; fax: +49 40/42803–5186; development and progression of diabetic nephropathy, a
e-mail: Wolf@uke.uni-hamburg.de
comprehensive understanding of the pathophysiology of this
Received 11 October 2004; accepted 21 October 2004 disease is mandatory [5]. Although initially thought that renal

© 2004 Blackwell Publishing Ltd


786 G. Wolf

injury in diabetic nephropathy is mainly caused by haemo- Table 1 Some of the genes implicated in the susceptibility and /or
dynamic alliterations such as hyperfiltration and hyper- progression of diabetic nephropathy (modified after 12)
perfusion, there is now clear evidence that these changes are
only one aspect of a complex series of pathophysiological Gene Gene variant
alterations caused by disturbed glucose homeostasis. The Promoter of RAGE 63-bp deletion (decreased risk)
last few years have provided a better insight into the complex Histocompatibility antigen DR3/ 4
pathophysiology of diabetic nephropathy on a molecular level. Angiotensin-converting enzyme D/ I
Angiotensinogen M235T
Aldose reductase Z + 2 alleles
Transforming growth factor β1 Leu10Pro, Arg25Pro
Are there different mechanisms operative in Apolipoprotein E e2 allele
nephropathy caused by type 1 or 2 diabetes? Paraoxonase 1 T107C, Leu54Met
Interleukin 1β T105C
Atrial natriuretic peptide C708T
Although debated for some time that diabetic nephropathies
Glucose transporter 1 Xba1/ HacIII
owing to either type 1 or 2 diabetes are specific entities, Mannose-binding lectin YA/ YA, XA/ YA
there is now convincing evidence that basic pathophysio-
logical mechanisms eventually leading to nephropathy are
similar in type 1 and 2 diabetes [2]. However, in type 2
diabetes other noxious factors, being related or not related heterogeneity and by only small effects in limited cases in
to diabetes, such as hypertension, obesity, dyslipidaemia most studies (often < 200 cases).
and ischaemic renal disease caused by arteriosclerosis, could More recently, genome scan data become available for
additionally injure the kidney resulting in complex patterns diabetic nephropathy. This method allows for a comprehen-
of nephropathy. The pathophysiological changes before the sive genetic survey of the entire genome for chromosomal
development of type 2 diabetes have been classified as the regions that are linked with a specific trait, in this case
metabolic syndrome [3]. This metabolic syndrome may diabetic nephropathy. A recent genome scan for diabetic
additionally harm the kidneys through hyperuricaemia and nephropathy in African Americans identified susceptibility
obesity itself independently of hyperglycaemia [6,7]. loci on chromosomes 3q, 7p and 18q [14]. Another scan in
The risk of nephropathy is strongly determined by genetic Pima Indians also identified linkage to diabetic nephropathy
factors and only approximately 40 –50% of patients with on chromosome 7 [15]. This powerful method may in the
either type 1 or type 2 diabetes will ultimately develop neph- future more clearly identify the genetic risk to develop diabetic
ropathy [8]. Experimental data support this observation. nephropathy.
A recent study demonstrated that phenotypic changes in
bone marrow-derived mesangial cell progenitors transmit
diabetic nephropathy from donors with type 2 diabetes (db/
db mice) to naive, normoglycaemic recipients [9]. Genetic Haemodynamic changes
factors may directly influence the development of diabetic
nephropathy and /or may be clustered with genes influencing Glomerular haemodynamic changes including hyperfiltra-
other cardiovascular diseases [8]. There is ongoing research tion and hyperperfusion have been viewed as pivotal in the
in identifying genetic loci for diabetic nephropathy suscep- development of diabetic nephropathy and are found very
tibility through genomic screening and candidate gene early in the disease process [16]. Principally, an elevation
approaches [10–12]. Although some potential genes have in glomerular capillary pressure causing an enhanced trans-
been identified, linkage was only present in defined ethnic capillary hydraulic pressure gradient as well as an increase
subpopulations and not in the majority of patients. An in glomerular plasma flow have been observed. These
incomplete list of previously implicated genes is shown in changes result from a decrease in both afferent and efferent
Table 1. The major problem with such studies is a non- arteriolar resistance, whereas the former is more dilated
simplistic mendelian inheritance mode with several genes than the latter leading to the increased glomerular capillary
likely involved [12]. A potential association between poly- pressure [16]. Formally, these changes could be described
morphisms in candidate genes and the development and as a defect in autoregulation because in the normal situation
progression of nephropathy has been widely studied. These an increase in perfusion pressure would result in preglo-
case–control retrospective studies are often problematic and merular vasoconstriction in order to maintain glomerular
clear guidelines for such polymorphism studies have been filtration rate at a constant rate [17]. Many diverse factors
provided [13]. The complexity of this genetic linkage analysis including prostanoids, nitrogen oxide (NO), atrial natriu-
is exemplified by various studies with controversial results retic factor, growth hormone, glucagon, insulin, angiotensin
investigating the insertion or deletion polymorphism of the II (ANG II), and others have been implicated as agents
angiotensin-converting enzyme (ACE). The current opinion causing hyperperfusion and hyperfiltration [16]. Elevated
is that the ACE polymorphism is, at best, in certain ethnic intraglomerular pressure has been linked to an increase
population associated with progression of disease but not in mesangial cell matrix production and thickening of the
as a predictor of the development of diabetic nephropathy glomerular basement membrane, eventually leading to
[8]. These discrepancies could be easily explained by genetic glomerulosclerosis [18,19]. There has been a discussion

© 2004 Blackwell Publishing Ltd, European Journal of Clinical Investigation, 34, 785–796
Diabetic nephropathy 787

going on for years about whether haemodynamic or struc- Table 2 Renal structural abnormalities found in diabetic
tural changes are more important for the manifestation of nephropathy
diabetic nephropathy [16]. However, it is now clear that
these processes are intimately interwoven. For example, Mesangial expansion
high glucose stimulates the synthesis of ANG II, which itself Glomerulosclerosis (diffuse, nodular)
exerts haemodynamic as well as trophic, inflammatory and Fibrin cap lesion
profibrogenic effects on renal cells [18,20]. Other factors Capsular drop lesion
Basement membrane thickening (glomerular and tubular)
induced by the diabetic environment that could influence
Endothelial foam cells
glomerular haemodynamics are vascular endothelial growth Podocyte abnormalities
factors (VEGFs), and recent evidence indicates that even Armanni-Ebstein cells (proximal tubules stuffed with glycogen)
cytokines, such as transforming growth factor beta (TGF- Tubular atrophy
β), may mediate hyperfiltration by dilatation of the vas affe- Interstitial inflammation
rens by inhibiting calcium transients [20]. Furthermore, Interstitial fibrosis
TGF-β increases NO production in early diabetes, probably Arteriosclerosis
by up-regulation of endothelial NO synthase (eNOS) mRNA
expression and by enhancing arginine resynthesis [21].Thus,
TGF-β could clearly play a role in diabetic vascular dys- diffuse intercapillary glomerulosclerosis. The earliest mor-
function and may mediate some of the haemodynamic phological change of diabetic nephropathy is expansion of
changes associated with early diabetic nephropathy [22]. the mesangial area [26]. This is caused by an increase in
On the other hand, shear stress and mechanical strain, extracellular matrix deposition and mesangial cell hyper-
resulting from altered glomerular haemodynamics, induce trophy. Structural–functional relationship investigations
the autocrine and/or paracrine release of cytokines and indicate a highly significant inverse correlation between
growth factors [18]. For example, exposing mesangial, glomerular filtration rate and mesangial expansion [26].
glomerular endothelial or podocytes to shear stress induces We have intensively studied the molecular mechanisms of
specific cellular responses including activation of certain how hyperglycaemia induces mesangial hypertrophy [29 –
signal transduction systems, growth responses, enhanced 33]. After a short period of proliferation, mesangial cells
synthesis of hormones and cytokines (e.g. ANG II, TGF-β), exposed to high glucose become arrested in the G1-phase
and increased production of extracellular matrix proteins of the cell cycle [30]. This G1-phase arrest is mediated by
[16]. These findings suggest that local haemodynamic stress p27Kip1, an inhibitor of cyclin-dependent kinases [30,31].
contributes to the structural changes of diabetic nephro- High glucose via activation of mitogen-activated protein
pathy by the local activation of cytokines and growth factors. kinases (MAPKs) lead to a post-transcriptional increase
As an alternative explanation, a primary abnormality in in p27Kip1 expression [33]. Deletion of the p27Kip1 gene
sodium reabsorption has been linked to glomerular hyper- attenuates high glucose-induced hypertrophy of mesangial
filtration in diabetic nephropathy [23]. This explanation cells [32]. In addition, ANG II further enhances p27Kip1
suggests that an increase in reabsorption of sodium chloride induction and blockade of ANG II attenuates high glucose-
in proximal tubules or loops of Henle leads to an increase mediated mesangial cell hypertrophy [31].
in the glomerular filtration rate by an intact macula-densa Thickening of the glomerular basement membrane
mechanism [23]. Diabetes-induced hypertrophy of tubules (GBM) occurs early and is already found 1 year after onset
that mediate stimulated sodium chloride reabsorption could of type 1 diabetes [34]. As shown in Fig. 1, thickening of the
be pivotal in this process, linking again structural changes GBM is progressive over years; both increased extracellular
with haemodynamic adaptation in diabetic nephropathy matrix synthesis and impaired removal contribute to GBM
[20]. thickening. Several biochemical alterations of the GBM
occur in diabetic nephropathy [34]. There is an increase in
collagen type IV deposition, whereas the expression of heparan
sulphate and the extent of sulphation decreases. In contrast
Structural abnormalities to the mesangial matrix in which the α1 and α2 of type IV
collagen are mainly expressed, the GBM contains α3, α4,
It is quite obvious that a systemic disease like diabetes and α5 chains [35,36]. In diabetic nephropathy, there is an
mellitus could result in injury of all renal compartments up-regulation of α1(IV) and α2(IV) chains in mesangial
[Table 2]. Consequently, glomerulosclerosis, vascular cells, whereas α3(IV) and α4 (IV) expression is increased
diseases and changes of the tubulointerstitial architecture in the GBM [36]. Deposition of collagen type I and III in
with tubular atrophy and interstitial fibrosis have all been the mesangial area occurs late in glomerulosclerosis and is
described in diabetic nephropathy [24 –28]. Since the original not an early event [35].
description by Kimmelstiel and Wilson, nodular glomeru- Glomerular epithelial cells (podocytes) directly cover the
losclerosis has been considered as a hallmark of diabetic GBM and there is recent evidence that alterations in struc-
nephropathy [24]. However, neither is this lesion specific ture and function of podocytes occur early in diabetic
for diabetic nephropathy because similar changes could be nephropathy [37–39]. Podocytes adhere to the GBM by
also detected in light-chain nephropathy nor is it the most α3β1 and α2β1 integrins. Hyperglycaemia induces a dys-
frequent glomerular histological abnormality which is regulation in integrin expression, influencing the interaction

© 2004 Blackwell Publishing Ltd, European Journal of Clinical Investigation, 34, 785–796
788 G. Wolf

Figure 1 Renal biopsies (electron-microscopy) from different patients suffering for various periods (3 month up to 12 years) from type
1 diabetes. There is a progressive increase in the thickness of the glomerular basement membrane from normal (left panel) until narrowing
of capillary loops and massive expansion of the mesangium by extracellular matrix (right panel). The process takes years until the right
stage is reached. Courtesy of Professor Helmchen, Institute of Pathology, University of Hamburg.

between podocytes and the GBM [35]. Longitudinal stud-


ies in humans with diabetic nephropathy demonstrated a
reduction in podocyte number during follow up which
closely correlated with proteinuria [37]. For example, Steffes
et al. determined the glomerular cell number with modern
morphometric techniques in patients with type 1 diabetes
and compared those numbers with age-matched normal
individuals [38]. They found a reduction in the podocyte
number in diabetic patients of all ages, with reduced podo-
cytes per glomerulus even in diabetes of short duration [38].
In addition, renal biopsies from Pima Indians with type 2
diabetes showed a broadening in podocyte foot processes
Figure 2 Reactive oxygen species (ROS) as a common mediator
and a concomitant reduction in the number of podocytes
of pathophysiological effects of hyperglycaemia. Increased uptake
per glomerulus [39]. In an European collective of White of glucose into cells leads to stimulated mitochondrial ROS
type 2 diabetics, Vestra et al. found a significant reduction formation. This oxidative stress, in turn, activates different
in the numerical density of podocytes per glomerulus in processes involving protein kinase C (PKC), NF-κB, cytokines,
patients with type 2 diabetes that were normoalbuminuric formation of advanced glycation end-products (AGEs), and others.
[40]. Abnormalities in podocyte structure are also observed
in animal models of diabetic nephropathy. Renal nephro-
pathy of Zucker fa/fa rats, a model of type 2 diabetes with way [Fig. 2]. All of these pathways could reflect a single
ultimate development of segmental glomerulosclerosis, starts hyperglycaemia-induced process of overproduction of reactive
with damage to podocytes, including foot process efface- oxygen species (ROS). Hyperglycaemia leads to an increase
ment and cytoplasmic accumulation of lipid droplets [41]. in mitochondrial ROS formation [43]. The first step in this
Early progressive podocyte damage antedates the develop- process is the transport of glucose into the cells through
ment of glomerulosclerosis and tubulointerstitial damage in specific glucose transporters [Fig. 3]. Mesangial cells express
this model [42]. insulin-sensitive glucose transporters (GLUT-4) as well as
a brain type of glucose transporters (GLUT-1) through
which excessive extracellular glucose could easily enter the
cell in an insulin-independent manner [44,45]. The role of
Oxidative stress as a common mediator GLUT-1 was clarified by overexpression of this transporter
in mesangial cells in which a stimulated production of
On a molecular level, at least five major pathways have been extracellular matrix proteins was then detected even under
implicated in glucose-mediated vascular and renal damage: normal external glucose concentrations [45]. An increase in
increased polyol pathway flux, increased hexosamine pathway glucose uptake leads to overproduction of electron donors
flux, activation of NF-κB, increased advanced glycation (NADH and FADH2) from stimulated glycolysis and the
end-product (AGE) formation, stimulation of ANG II tricarboxylic acid cycle [43]. At the mitochondrial inner
synthesis and activation of the protein kinase C (PKC) path- membrane, where the electron-transport chain is localized,

© 2004 Blackwell Publishing Ltd, European Journal of Clinical Investigation, 34, 785–796
Diabetic nephropathy 789

of glucose to sorbitol requires NADPH-depleting cells of an


important substrate for the regeneration of glutathione. As
a consequence, this exacerbates intracellular oxidative stress.
Second, the intermediate 3-deoxyglucone is a precursor of
AGEs. Thus, activation of the polyol pathway increases oxi-
dative stress and also enhances AGE formation. Interestingly,
recent data suggest that the calcium antagonist amlodipine
inhibits sorbitol formation, indicating blockade of the polyol
pathway [49].
An increased flux of glucose through the hexosamine
pathway has also been linked to mechanisms of diabetic
nephropathy, particularly an increase in TGF-β [50].
Fructose-6-phosphate from glycolysis is converted to
glucosamine-6-phosphate in this pathway. Glycosylation of a
transcription factor such as Sp1 by N-acetylglucosamine
Figure 3 Overview of processes leading to the generation of stimulates TGF-β transcription. In addition, an increase in flux
reactive oxygen species (ROS). Glucose is taken up into the cells through the hexosamine pathway up-regulates the expression
through insulin-sensitive glucose transporters (GLUT-4) that are of up-stream stimulatory factors (USFs) which transactivate
not regulated by insulin. Therefore, hyperglycaemia leads to an the TGF-β1 promoter [51].
increase in the intracellular glucose concentration, which is
Intracellular accumulation of glucose also increases de
subsequently metabolized to pyruvate through glycolysis. During
novo formation of diacylglycerol (DAG) from glycolytic
this process NADH is generated, which is transported into
mitochondria. In the tricarboxylic acid cycle (TCA) pyruvate is intermediates such as dihydroxyacetone phosphate [44]. An
further metabolized to CO2 and water with the generation of increase in DAG activates several isoforms of PKC. Inhibi-
additional electron donors (NADH and FADH2). This increase in tion of PKC-β, the major isoform induced in the kidney by
electron donors leads to a disturbance of the respiration chain with hyperglycaemia, ameliorates diabetic nephropathy. More-
the formation of ROS. Details compare with text. over, activation of PKC could, in turn, further stimulate
MAPKs. Erk 1,2 as well as p38 MAPK have been impli-
cated as signalling intermediates in diabetic nephropathy
the increase in electron donors (NADH, FADH2) generates [44]. MAPKs are additionally activated by ROS and there
a high membrane potential by pumping protons across the is likely cross-talk between the various pathways [44]. The
inner membrane [Fig. 3]. As a consequence, electron importance of PKC in the development of some changes
transport is inhibited at complex III increasing the half-life of diabetic nephropathy is underscored by recent studies
of free-radical intermediates of coenzyme Q, which finally demonstrating that albuminuria was absent in diabetic

reduces O2 to superoxide ( O2 ). Experimental studies with PKC-α knockout mice [52]. However, glomerular hyper-
endothelial cells have demonstrated that overexpression of trophy or the up-regulation of TGF-β was not influenced
manganese superoxide dismutase (MnSOD) abolished by the lack of PKC-α [52].
the hyperglycaemia-induced generation of ROS, and over-
expression of uncoupling protein-1 (UCP-1) collapsed the
proton electrochemical gradient and prevented generation
of ROS [43]. Furthermore, UCP-1 and MnSOD pre- Advanced glycation end-products
vented, in this system, high glucose-activation of the polyol
pathway, stimulated AGE formation and increased PKC Advanced glycation end-products (AGEs) are a complex
activation, suggesting a pivotal role of mitochondrial- and heterogeneous group of compounds implicated in
generated ROS in these processes [43]. diabetes-related complications including nephropathy [53].
The formation of ROS does not only occur in mitochon- Glucose reacts nonenzymatically with amino groups in pro-
dria. Recent evidence suggests that the 12-lipoxygenase teins, nuclei acids and lipids through a series of steps to form
pathway of arachidonic acid metabolism is activated by high Schiff bases and Amadori products to finally produce AGEs
glucose in podocytes [46]. Stimulation of 12-lipoxygenase [54]. These processes occur over a period of weeks, thereby

generates O2 , making this an important additional path- affecting long-lived proteins.Therefore, structural components
way of ROS formation independent of mitochondria [47]. of tissue matrix such basement membranes are important
Finally, a reduction of antioxidants such as glutathione targets [55]. For example, glycation inhibits the interactions
contributes to oxidative stress in the diabetic state [48]. required for self-assembly of type IV collagen and laminin
Glucose is converted to sorbitol by aldose reductase and [56]. Glycation of proteins can be accompanied by oxida-
subsequently to fructose by sorbitol dehydrogenase in the tion, a process called glycoxidation, which is obviously
polyol pathway. An increase in cellular glucose uptake will enhanced by oxidative stress [57,58]. Some of the better
shift some of the glucose into this pathway. The rate of sor- characterized AGEs such as pentosidine and N-carboxymethyl-
bitol production depends primarily on the intracellular lysine (CML) are examples of such glyoxidated products.
availability of glucose. Activation of the polyol pathway ANG II may further stimulate AGE formation. Interest-
could have several deleterious effects [44,48]. First, reduction ingly, ACE inhibitors and AT1-receptor antagonist inhibit

© 2004 Blackwell Publishing Ltd, European Journal of Clinical Investigation, 34, 785–796
790 G. Wolf

in vitro CML and pentosidine formation by interfering mRNA and protein in renal tubular and mesangial cells in
with various oxidative steps, including carbon-centred and vitro and in vivo [71–74]. Glomerular hypertension is an
hydroxyl radical [59]. Furthermore, products of the polyol additional potent stimulus for renal TGF-β expression link-
pathways forming intermediate products such as 3- ing metabolic and haemodynamic effects [68]. TGF-β and
deoxyglucosone could further induce nonoxidative AGEs its type II receptor expression are increased in models of
(deoxyglucasone-lysine dimer). Although the majority of type 1 and 2 diabetes [75]. Finally, up-regulation of the
AGEs is usually endogenously formed, it is apparent that TGF-β system and production of TGF-β with a release in
exogenous AGEs from food intake may contribute to the the systemic circulation has been found in humans with
overall burden, particularly in situations with reduced renal diabetes (for a review see [68]). A consensus nucleotide
function [55]. In early diabetic nephropathy, CML and sequence termed the ‘glucose response element’ has been
pentosidine accumulate in the expanded mesangial area and characterized in the TGF-β promoter [73]. Activation of
the thickened glomerular capillary wall [57]. The receptor for PKC and p38 MAP kinase plays an additional role through
AGEs, called RAGE, is a multiligand member of the AP-1 binding sites in transcriptional stimulation of the
immunoglobulin family of cell-surface receptors [60]. In TGF-β gene [73]. Additionally, a post-transcriptional acti-
addition, other moieties such as galectin-3, CD36, macro- vation of latent to active TGF-β is induced in mesangial cells
phage scavenger receptor and others bind AGEs [58]. through thrombospondin-1 [74]. TGF-β stimulates the pro-
RAGE is the signal transduction receptor for AGEs and is duction of several extracellular matrix proteins including
widely expressed on various renal cell types. Stimulation of fibronectin, and types I, III, and IV collagens [72]. TGF-β
this receptor causes expression of proinflammatory cytokines also inhibits the degradation of extracellular matrix com-
through NF-κB activation [58]. Besides induction of ponents through inhibition of matrix metalloproteinases.
cytokines, RAGE directly contributes to inflammation by Long-term treatment of mice with either type 1 or 2 diabetes
serving as an endothelial adhesion receptor promoting and nephropathy with neutralizing anti-TGF-β antibodies
leucocyte recruitment [61]. AGEs also induce TGF-β as a prevented mesangial matrix expansion and attenuated the
profibrogenic cytokine [58]. AGEs are filtered by glomeruli decrease in renal function [75]. Bone morphogenic protein
and are reabsorbed by proximal tubules [62]. Although not (BMP)-7, a member of the TGF-β superfamily playing a
directly binding to AGEs, megalin is involved in the tubular major role during embryonic development, can antagonize
uptake of filtered AGEs [62]. In tubular cells, RAGE acti- TGF-β-dependent fibrogenesis in renal cells [76].
vation leads to transition of these cells into myofibroblasts; Connective tissue growth factor (CTGF) is one of the
a response involved in the development of tubular atrophy more recently characterized growth factors and is a 36–38-
and interstitial fibrosis of ongoing diabetic nephropathy kDa cystein-rich peptide [77]. It may in fact be the direct
[63]. RAGE expression itself is up-regulated in the diabetic down-stream mediator of some effects hitherto thought to
state, for example on podocytes. A novel therapeutic con- be caused by TGF-β [66,77]. Further studies such as inter-
cept is the application of soluble RAGE (sRAGE), which ference with CTGF expression and /or receptor blockade are
scavenges and neutralizes AGE [58]. In addition to being ultimately necessary to test its role in diabetic nephropathy.
a key player in the development of diabetic nephropathy, Vascular endothelial growth factor is an approximately
AGEs also modify essential functions of leucocytes and con- 34–46-kDa homodimeric glycoprotein which exists in at
tribute to the reduced immune function observed in diabetes least five different isoforms produced by differential exon
mellitus [64]. splicing [69]. Vascular endothelial growth factor itself is
expressed in podocytes, distal tubules and the collecting duct
[69,78]. It has been speculated that heavier VEGF isoforms
may be carried across the glomerular basement membrane
Cytokines and growth factors exerting than their effects on endothelial cells. Extracellular
matrix proteins regulate VEGF transcription in podocytes [79].
Various growth factors, cytokines, chemokines and vaso- Circulating VEGF concentrations are significantly increased
active agents have been implicated in structural changes of in type 1 and 2 diabetes. Several studies indicate that VEGF
diabetic nephropathy [65 – 67]. A comprehensive review of mRNA and protein expression are enhanced in kidneys with
all growth factors potentially involved in diabetic nephro- early diabetic nephropathy (for a review see [69]). Furthermore,
pathy is beyond the scope of this article and excellent reviews AGEs contribute to expression of VEGF. A functional role
exist [65–69]. Insulin-like growth factors (IGFs) are among of VEGF in diabetic nephropathy was demonstrated by the
the most widely and earliest studied growth factors in dia- observation that monoclonal anti-VEGF antibodies admin-
betic nephropathy but the exact role remains elusive [70]. istered to diabetic rats decreased hyperfiltration, albuminuria
An early and temporary increase in renal IGF-I protein after and glomerular hypertrophy in these animals [80].
the onset of diabetes is found in various animal models and
this increase is caused by hyperglycaemia [70]. Interference
with the IGF-I axis partly attenuates diabetic nephropathy
in some models [65]. Hypoxia and diabetic nephropathy
A very important profibrogenic factor for the develop-
ment of diabetic nephropathy is TGF-β [68,71]. High Recent clinical studies have provided evidence that even
glucose, Amadori products as well as AGEs increase TGF-β mild anaemia (Hb < 13·8 g dL−1) increases the risk for

© 2004 Blackwell Publishing Ltd, European Journal of Clinical Investigation, 34, 785–796
Diabetic nephropathy 791

progression in patients with type 2 diabetes and nephropathy Pivotal role of the renin-angiotensin-aldosterone
[81]. Moreover, treating anaemia early in renal failure with system
erythropoietin slows the decline of renal function [82,83].
The exact mechanisms by which anaemia increases the risk Drugs interfering with the renin-angiotensin-aldosterone
for progression of diabetic nephropathy are incompletely system (RAAS) are a mainstay of therapy in preventing the
understood but a few suggestions could be made. Anaemia progression of diabetic nephropathy since the seminal
likely causes renal hypoxia [84]. It has been described in observation by Zatz and coworkers that an ACE-inhibitor
experimental models of chronic renal injury that hypoxia is prevented the development of nephropathy in experimen-
an important factor aggravating interstitial fibrosis, partly tally induced diabetes [94]. Although initially considered to
by the induction of factors such as TGF-β and VEGF [84]. act solely through normalization of systemic and glomerular
This induction of growth factors and cytokines is mediated hypertension, it is now clear that inhibition of the RAAS has
by hypoxia-inducing factor-1 (HIF-1), and ANG II can many effects, including antifibrotic and anti-inflammatory
further increase this important transcription factor [85]. On mechanisms [95]. In fact, ANG II itself induces in renal
the other hand, an erythropoietin application may have cells many proinflammatory and profibrogenic cytokines,
additional effects besides correcting anaemia, and the mobil- chemokines and growth factors [Fig. 4]. It has been dem-
ization of potential progenitor cells by this treatment has onstrated that many, if not all, RAAS components are
become the focus of active research [86]. Certainly, more present in the kidney, for example in tubular cells [95]. High
experiments are necessary to decipher how anaemia may glucose stimulates expression of renin and angiotensinogen
exactly contribute to the development and progression of in mesangial and tubular cells [96–99]. This stimulation
diabetic nephropathy but this topic will be of great interest. results in an increase in local ANG II concentrations which
may, in turn, through autocrine and paracrine pathways
induce a whole battery of different cytokines and growth
factors [95]. Experimental studies indicate that a high
Inflammation and diabetic nephropathy glucose-mediated generation of ROS is important in the up-
regulation of angiotensinogen in proximal tubular cells [98].
Biopsy studies from patients with diabetic nephropathy and Inhibition of the RAAS reduces proteinuria in diabetic
investigations in various animals models revealed the nephropathy. Angiotensin-converting enzyme inhibitor or
presence of inflammatory cells in glomerular and tubulo- AT1-receptor antagonist treatment attenuates podocyte
interstitial compartments [87]. Mononuclear cell infiltrates foot process broadening in rats with streptozotocin-induced
are often present [88]. Monocyte chemoattractant protein diabetes [100–103]. An AT1-receptor antagonist, but not
1-(MCP-1) is an important chemokine for macrophages/
monocytes. High glucose stimulates MCP-1 expression in
mesangial cells. Increases in tubular expression of MCP-1
and RANTES, another chemokine, have been found in
renal biopsies from patients with diabetic nephropathy and
were adjacent to infiltrating immune cells [88 – 92]. The
proinflammatory transcription factor NF-κB was detected
mainly in tubular cells in biopsy specimens from 11 patients
with type 2 diabetes and overt nephropathy, indicating that
proteinuria may have contributed to this activation [90].
Fractalkine expression is induced in renal endothelial cells
from diabetic rats [91]. This induction promotes strong
adhesion of T cells and monocytes to the endothelium via
the receptor CX3CR1 [91]. It appears that circulating levels
of chemokines such as MCP-1 are already enhanced in the
metabolic syndrome and the adipocyte could be an impor-
tant source [93]. The expression of proinflammatory mol-
ecules such as chemokines could be further stimulated in
podocytes and tubular cells by proteinuria acting in concert Figure 4 Summary of mediators involved in the pathophysiology
with hyperglycaemia and AGEs. As infiltrating mononuclear of diabetic nephropathy. High glucose leads to the generation of
cells release proteases and profibrogenic cytokines, includ- reactive oxygen species (ROS) and increases advanced glycation
ing TGF-β, such proinflammatory infiltrates contribute end-product (AGE) synthesis. Reactive oxygen species stimulate
local ANG II generation, which increases proteinuria, but
to the destruction of nephrons in diabetic nephropathy.
proteinuria also further enhances tubular angiotensin II (ANG II)
Consequently, treatment of diabetic animals with anti-
synthesis. Angiotensin II also increases AGE formation and is
inflammatory drugs such as mycophenolate mofetil prevents pivotal in the induction of various cytokines and growth factors.
the development of glomerular injury [92]. Diabetes- Some of these cytokines directly stimulate extracellular matrix
associated induction of proinflammatory cytokines is partly synthesis (e.g. TGF-β, CTGF), whereas other mediate
dependent on ANG II [88]. Therefore, inhibition of ANG II inflammation (MCP-1). As a consequence of these processes, tissue
exerts anti-inflammatory effects also. destruction and fibrosis will result.

© 2004 Blackwell Publishing Ltd, European Journal of Clinical Investigation, 34, 785–796
792 G. Wolf

the calcium-channel blocker amlodipine, normalized the receptor blocker (aldosterone escape phenomenon) is asso-
reduced nephrin expression in podocytes from spontane- ciated with a decline in glomerular filtration rate in patients
ously hypertensive rats with superimposed streptozotocin- with nephropathy owing to type 1 diabetes [113]. These data
induced diabetes [103]. Thus, a local increase in ANG II demonstrate that aldosterone contributes to the progression
leads to suppression of nephrin expression in podocytes. of diabetic nephropathy despite blockade of the AT1-receptor
Podocytes express AT1- and probably AT2-receptors after [113]. On the other hand, spironolactone decreases pro-
injury and could respond to a stimulation with ANG II teinuria in patients with 2 diabetes and early nephropathy
[104]. Transgenic rats with targeted overexpression of the [114]. As spironolactone is a nonspecific aldosterone recep-
AT1-receptor to podocytes showed pseudocysts in podo- tor antagonist that may also bind to other steroid receptors
cytes, followed by foot process effacement and local detach- explaining adverse sexual effects, novel antagonists (e.g.
ments [105]. These changes subsequently progressed to eplerenone) have been developed [115]. Preliminary studies
focal segmental glomerulosclerosis. It has been previously indicate that eplerenone decreases microalbuminuria in
shown that high ambient glucose concentrations induce patients with type 2 diabetes independent of its antihyper-
ANG II formation in podocytes through up-regulation of tensive effects [115]. In addition to direct effects on renal
angiotensinogen expression [106]. Furthermore, proteinu- cells, aldosterone also potentates ANG II-mediated signal
ria and a likely transit of proteins through the ultrafiltration processes such as MAPK activation, indicating that ANG
barrier activates ANG II formation in podocytes [106; II and aldosterone act in concert [16].
Fig. 4]. Finally, mechanical stretch could increase ANG II Taking into account the many effects of the RAAS in the
generation in podocytes [107]. Interestingly, ANG II pathophysiology of diabetic nephropathy, early treatment
formation as a consequence of mechanical stress appears to with drugs interfering with the RAAS are a necessary
be independent of ACE [107]. In this regard it has been prerequisite to prevent development and progression of
demonstrated that chymase, an ANG II-forming enzyme nephropathy in diabetes mellitus. This treatment has entered
not inhibited by ACE inhibitors, is up-regulated in glomer- clinical practice and should be part of the optimal manage-
uli of patients with nephropathy owing to type 2 diabetes ment of every diabetic patient.
[108]. This observation suggests that local glomerular
formation of ANG II in the diabetic state may be partly
independent of ACE and could not be abolished by ACE
inhibition alone. As a consequence, an increase in local Regression of diabetic nephropathy: a dream
ANG II concentrations in the podocyte microenvironment comes true?
suppresses nephrin expression, and thereby enhances ultra-
filtration of proteins which further contributes to podocyte Common wisdom suggests that the morphological altera-
damage. tions of advanced diabetic nephropathy (e.g. glomeruloscle-
Angiotensin II plays an important role in mediating pro- rosis, interstitial fibrosis) are not reversible and represent a
teinuria by various mechanisms including hyperfiltration, one-way road to end-stage renal disease. Recent clinical
opening of nonselective pores in the ultrafiltration barrier, studies show, however, that this pessimistic scenario is not
modifying the composition of the GBM and reducing nephrin necessarily true. Several studies indicate that aggressive
expression on podocytes (for a review see [18]). In addition, blood pressure control with agents that interfere with the
it has been found that ANG II increases tubular reabsorp- RAAS leads to remission of overt proteinuria (for a review
tion of ultrafiltered proteins, contributing thereby to tubular see [117]). High-dose ACE-inhibitor treatment induces
inflammation and eventually fibrosis [18]. A recent study regression in experimental nondiabetic models of glomer-
has somewhat questioned this straightforward explanation ulosclerosis [118]. Fioretto and colleagues performed a serial
because targeted deletion of the AT1A receptor in mice does renal biopsy in a collective of patients with type 1 diabetes
not protect from progressive renal injury induced by over- and nephropathy who received a pancreas transplant [119].
load proteinuria [109]. However, as it has been convincingly These investigators found that morphological alterations
demonstrated that the AT2-subtype of the ANG II-receptor of diabetic nephropathy including increased thickness of
could induce proinflammatory effects through NF-κB acti- basement membranes and mesangial expansion decreased
vation [110,111], both subtypes of receptors are probably 10 years after receiving the pancreas transplant. The data
involved in the deleterious effects of proteinuria on the clearly demonstrate that the control of blood glucose levels
tubulointerstitium. can reverse the development of diabetic nephropathy, but
Recent experimental evidence also indicates a role of this process takes time. In addition, Perkins et al. showed
aldosterone, working independently from ANG II, in the that the onset of microalbuminuria does not imply inexor-
development of diabetic nephropathy [99]. The aldosterone ably progressive nephropathy, at least not in patients with
antagonist spironolactone attenuates increased collagen type 1 diabetes [120]. This study provides indirect evidence
deposition in rats 3 weeks after streptozotocin administra- that aggressive management aiming at various risk factors
tion [99]. Spironolactone also suppresses the enhanced could lead to a remission of albuminuria. As no renal bio-
TGF-β1 expression in this model [99]. These findings have psies were performed in this study, it remains unproven
been extended to patients. In a preliminary study, Schjoedt whether morphological changes of diabetic nephropathy are
and colleagues observed that an increase in the plasma also improved [120]. Nervertheless, there is light at the end
aldosterone level during long-term treatment with an AT1- of the tunnel and a better understanding of the complex

© 2004 Blackwell Publishing Ltd, European Journal of Clinical Investigation, 34, 785–796
Diabetic nephropathy 793

pathophysiology of diabetic nephropathy will likely improve 18 Wolf G, Butzmann U, Wenzel UO. The renin-angiotensin
a multifactorial management to induce regression. system and progression of renal disease: from hemodynamics
to cell biology. Nephron Physiol 2003;93:P3–13.
19 Anderson S, Rennke HG, Brenner BW. Therapeutic
advantage of converting enzyme inhibitors in arresting
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