Kidney International, Vol. 58, Suppl. 77 (2000), pp.
S-93–S-98
Role of angiotensin II in diabetic nephropathy
DAVID J. LEEHEY, ASHOK K. SINGH, NAHID ALAVI, and REKHA SINGH
Hines Veterans Affairs Hospital, Hines, Illinois; Loyola University School of Medicine, Maywood, Illinois; Hektoen Institute for
Medical Research, Chicago, Illinois; and Westside Veterans Affairs Hospital, Chicago, Illinois, USA
Role of angiotensin II in diabetic nephropathy. Considerable
evidence suggests that the intrarenal renin-angiotensin system
plays an important role in diabetic nephropathy. Angioten-
sin-converting enzyme (ACE) inhibitors and angiotensin II
(Ang II) receptor blockers (ARBs) can attenuate progressive
glomerulosclerosis in disease models and can slow disease pro-
gression in humans. Because agents that interfere with Ang II
action may decrease glomerular injury without altering glomer-
ular pressures, it has been suggested that Ang II has direct
effects on glomerular cells to induce sclerosis independent of
its hemodynamic actions. To study nonhemodynamic effects
of Ang II on matrix metabolism, many investigators have used
cell culture systems. Glucose and Ang II have been shown to
produce similar effects on renal cells in culture. For instance,
incubation of mesangial cells in high-glucose media or in the
presence of Ang II stimulates matrix protein synthesis and
inhibits degradative enzyme (e.g., collagenase, plasmin) activ-
ity. Glucose and Ang II also can inhibit proximal tubule pro-
teinases. Glucose increases expression of the angiotensinogen
gene in proximal tubule cells and Ang II production in primary
mesangial cell culture, which indicates that high glucose itself
can activate the renin-angiotensin system. The effects of glu-
cose and Ang II on mesangial matrix metabolism may be medi-
ated by transforming growth factor-␤ (TGF-␤). Exposure of
mesangial cells to glucose or Ang II increases TGF-␤ expres-
sion and secretion. Their effects on matrix metabolism can be
blocked by anti-TGF-␤ antibody or ARBs such as losartan,
which also prevents the glucose-induced increment in TGF-␤
secretion. Taken together, these findings support the hypothe-
sis that the high-glucose milieu of diabetes increases Ang II
production by renal, and especially, mesangial cells, which re-
sults in stimulation of TGF-␤1 secretion, leading to increased
synthesis and decreased degradation of matrix proteins, thus
producing matrix accumulation. This may be an important
mechanism linking hyperglycemia and Ang II in the pathogene-
sis of diabetic nephropathy.
Diabetic nephropathy is characterized by expansion
of extracellular matrix (ECM) in the glomeruli, which
eventually leads to proteinuria and renal failure. Accu-
mulation of ECM components such as collagen has been
demonstrated in diabetic glomeruli [1–3] and in mesan-
gial cells incubated in high glucose media [4–6]. Both
increased synthesis [4–8] and decreased degradation
Key words: diabetes, angiotensin, glucose, matrix.
 2000 by the International Society of Nephrology
S-93
CYTOKINES AND GROWTH FACTORS
[8–15] of matrix components are responsible for matrix
accumulation.
Mediators of mesangial matrix expansion in diabetic
nephropathy have not been fully identified. A prominent
role for the peptide angiotensin II (Ang II) has been
suggested by experimental and clinical evidence indicat-
ing that angiotensin-converting enzyme (ACE) inhibi-
tors and angiotensin receptor blockers (ARBs) have re-
noprotective effects that cannot be entirely explained by
their blood pressure lowering effects [16–19]. The ACE
inhibitor enalapril inhibits gene expression of ECM pro-
teins in diabetic rats [20], suggesting that Ang II is impor-
tant in the development of glomerulosclerosis and that
preventing its formation can decrease matrix synthesis.
However, in studies in whole animals or humans, it is
difficult to separate the effects of blood pressure reduc-
tion from other biochemical effects of these agents.
Use of cell culture obviates these problems because
it allows separation of hemodynamic and nonhemody-
namic actions of Ang II. Studies using cell culture sys-
tems have provided data indicating that glucose and Ang
II can both directly increase matrix synthesis and de-
crease its degradation, and that Ang II inhibitors can
decrease glucose-induced matrix accumulation (see sub-
sequent discussion).
RENIN-ANGIOTENSIN SYSTEM IN
DIABETIC NEPHROPATHY
The status of the renin-angiotensin system (RAS) has
been extensively studied in diabetes. Earlier studies fo-
cused on the systemic RAS, and the data obtained have
been conflicting, with stimulation, suppression, and no
change in the system being reported [21, 22]. The multi-
tude of factors that influence the systemic RAS in addi-
tion to the different stages of disease and species studied
may explain many of these divergent findings. However,
in various diabetic models, increased renal renin content
relative to plasma renin levels has generally been found,
thus suggesting impaired renal renin release into the
circulation [22]. Decreased plasma renin activity is fre-
quently observed in clinical diabetic nephropathy, which
S-94 Leehey et al: Angiotensin in diabetic nephropathy
may be due to nonenzymatic glycation of prorenin with
decreased conversion to active renin [23]. Thus, diabetic
nephropathy has traditionally been considered a “low
renin” state. However, plasma renin activity may not
accurately reflect activity of the RAS in the kidney. An-
other problem has been the difficulty of accurate mea-
surement of plasma Ang II, which is an important issue
because discordance can exist between plasma renin and
Ang II levels [24].
More recently, the intrarenal RAS has been the focus
of extensive study. Abundant evidence indicates the exis-
tence of local tissue RASs that are regulated indepen-
dently of plasma RAS. Ballerman et al. [25] first reported
a decrease in glomerular Ang II receptors in the diabetic
rat 3 to 4 weeks after induction of the disease. Downregu-
lation of glomerular Ang II receptors implies that intra-
renal Ang II generation may be increased. The density
of Ang II receptors in the proximal tubules was reported
to be reduced in diabetic rats and was accompanied by
decreased mRNA expression for the AT1 receptor [26].
Recently, AT1 receptor density has also been shown to
be down-regulated in mesangial cells incubated in high-
glucose media [27]. Whole kidney ACE activity is low
in diabetes; however, this is probably due primarily to
proximal tubule suppression because staining for ACE
has been found to be enhanced in glomeruli and vascula-
ture of diabetic rats [28] and in patients with diabetic
nephropathy [29]. These data suggest that the term “in-
trarenal” RAS is an oversimplification, inasmuch as the
vascular RAS (vessels and glomeruli) appears to be regu-
lated differently from the tubulointestitial RAS. Renal
vasodilation in response to ARBs is enhanced in patients
with diabetes (despite the presence of low plasma renin
activity), again supporting the concept that the renal
vascular RAS is activated despite suppression of the
circulating RAS (abstract; Price et al, J Am Soc Nephrol
7:1363, 1996).
Ang II concentrations in several intrarenal compart-
ments including the glomeruli have been found to be
several orders of magnitude higher than those found
systemically [30]. This supports both the existence of a
local RAS acting independently of the systemic RAS
and also is consistent with the finding that in most renal
cell culture studies, effects of Ang II are observed at sub-
stantially higher concentrations (about 0.01–1.0 ␮mol/L)
than those found in the systemic circulation.
POTENTIAL MECHANISMS OF
ANG II-INDUCED RENAL INJURY
Ang II has many physiologic and biochemical effects
that could contribute to diabetic nephropathy (Table 1),
and thus the renoprotective effect of blocking the RAS
undoubtedly results from various mechanisms. The greater
antiproteinuric effects of ACE inhibitors and ARBs com-
pared to other antihypertensive agents may be explained
by greater lowering of glomerular capillary pressure with
these agents [31]. However, other mechanisms are proba-
bly involved in their renoprotective effects. Ang II can
directly increase cytokine formation and matrix accumu-
lation and stimulates growth and protooncogenes in re-
nal cells [32]. An increase in proteinuria due to Ang II
may itself lead to accelerated tubulointerstitial injury
[33]. Ang II has recently been demonstrated to increase
mesangial cell superoxide production and thus may exert
oxidant-induced renal injury (discussed later).
Effects of glucose and mechanical strain on
intrarenal RAS
Cultured glomeruli contain the substrate and enzymes
needed for Ang II generation [34]. Moreover, recent data
support the concept that the mesangial cells themselves
contain all of the elements of the RAS, including renin,
angiotensinogen, type I angiotensin II (AT1) receptors,
and ACE [35, 36]. Stretch/relaxation of cultured mesan-
gial cells has been shown to increase angiotensinogen
gene expression and production and increase AT1 recep-
tor expression [35]. Thus, not only may Ang II-induced
hemodynamic changes affect mesangial function but me-
chanical stress per se can increase the activity of the
mesangial RAS. Ang II is produced by primary cultures
of rat mesangial cells and glucose increases Ang II syn-
thesis by these cells [37] (Fig. 1). The mechanism by
which glucose increases mesangial Ang II production is
currently being studied in our laboratory. A possible
mechanism may be stimulation of angiotensinogen gene
expression, as has been demonstrated in tubular cells
[38]. The ability of mesangial cells to synthesize Ang II
may not require ACE, because angiotensin I formed
from cleavage of angiotensinogen may be converted to
Ang II through other peptidases. Thus, in diabetic cell
culture models, both mechanical and biochemical (i.e.,
high glucose) perturbations can increase the activity of
the intrarenal RAS.
Effects of glucose and Ang II on matrix metabolism
Glucose and Ang II have been shown to have similar
effects on glomerular cells in culture. For instance, incu-
bation of mesangial cells in high glucose media results
in stimulation of matrix protein synthesis [4–6, 8]. Ang
II has similar effects [39–41]. Both glucose loading [12]
and Ang II [37] inhibit mesangial cell collagenase activ-
ity, and these effects are blocked by losartan (Fig. 2).
Glucose-induced collagen accumulation in mesangial
cells is also blocked by losartan [37]. Ang II has been
demonstrated to increase plasminogen activator inhibi-
tor synthesis and decrease plasminogen activator activity.
These alterations in the plasminogen/plasmin system
would lead to decreased matrix degradation and in-
creased accumulation, both by the direct effects of plas-
min and through plasmin-mediated collagenase activa-
tion [42, 43]. In proximal tubule cells, glucose and Ang
II also both inhibit proteinase activity [44, 45].
 Leehey et al: Angiotensin in diabetic nephropathy
Table 1. Proposed mechanisms of angiotensin II effects in diabetic nephropathy
Hemodynamic effects
Systemic hypertension
Systemic and renal vasoconstriction
Increased glomerular capillary pressure and permeability
Mesangial cell contraction leading to reduction in filtration surface area
Fig. 1. Angiotensin II production by mesangial cells incubated with
varying concentrations of glucose (5 to 60 mmol/L) for 24 hours. Angio-
tensin II levels were measured in cell extracts by ELISA. Data shown
are the means ⫾ SEM of four experiments. Glucose loading increased
Ang II production in a concentration-dependent manner (*P ⬍ 0.05
vs. 5 mmol/L glucose). (From [37]).
Glucose and Ang II probably use similar signal trans-
duction pathways in renal cells in culture. Glucose stimu-
lates de novo synthesis of diacylglycerol (DAG) gener-
ated from glycolytic intermediates through the polyol
pathway; elevated DAG then leads to activation of pro-
tein kinase C (PKC), which then increases TGF-␤1 syn-
thesis and matrix protein synthesis in mesangial and tu-
bular cells. Ang II activates PKC through the AT1a
receptor. Activation of PKC probably thus occurs both
as a direct effect of glucose and by activation of AT1a
receptors. In addition, additive effects of high glucose
and Ang II on PKC activation may occur [21].
Effects of glucose and Ang II on TGF-␤ in
diabetic nephropathy
The cytokine TGF-␤ is known to be an important
mediator of matrix accumulation and fibrosis [46]. Glo-
meruli from diabetic animals and humans have increased
mRNA for TGF-␤ as well as other growth-promoting
S-95
Non-hemodynamic effects
Induction of renal hypertrophy and cell proliferation
Stimulation of extracellular matrix synthesis
Inhibition of extracellular matrix degradation
Stimulation of cytokine (e.g., TGF-␤, VEGF, endothelin) production
Stimulation of superoxide production
factors [47, 48]. It is therefore likely that TGF-␤ is in-
volved in the pathogenesis of diabetic nephropathy. In-
deed, the effects of glucose and Ang II on mesangial
matrix metabolism may be mediated by TGF-␤. Expo-
sure to high glucose results in an increase in TGF-␤
mRNA and secretion in mesangial cells [49, 50]. The
effects of elevated glucose on collagen synthesis can be
blocked by anti-TGF-␤1 antibody [51]. Decreased colla-
genase activity in rat mesangial cells after glucose loading
can also be reversed by anti-TGF-␤1 antibody, sug-
gesting that this cytokine also affects mesangial matrix
degradative mechanisms (Singh et al, Exp Nephrol, in
press). We have found that exogenous TGF-␤1 decreases
matrix metalloproteinase-2 levels and increases tissue
inhibitor of metalloproteinase-2 levels in rat mesangial
cells (Singh et al, Exp Nephrol, in press). Ang II stimu-
lates mesangial matrix synthesis, an effect mediated by
TGF-␤ [39]. The Ang II competitive inhibitor saralasin
prevented the effects of Ang II [39]. Similar effects of
Ang II on TGF-␤ synthesis also occur in proximal tubule
cells [52]. In rat mesangial cells, glucose-induced in-
creased TGF-␤1 secretion is blocked by the Ang II antag-
onist losartan [37] (Fig. 3). In a study employing AT1a
receptor–deficient mice made diabetic with streptozo-
tocin induction, increased renal TGF-␤ mRNA levels
were seen only in mice with an intact renin-angiotensin
system and not in the absence of the AT1a receptor (ab-
stract, Hutchison FN et al, J Am Soc Nephrol, 7:1872–
1873, 1996), further supporting the hypothesis that the
increase in TGF-␤ with diabetes is mediated by Ang II.
Taken together, these data suggest that the effects of
glucose in renal (and especially, mesangial) cells may be
mediated by stimulation of Ang II production, which
then leads to TGF-␤–mediated increased matrix synthe-
sis and decreased matrix degradation (Fig. 4).
Ang II-induced vascular growth factor expression
Vascular endothelial growth factor (VEGF), also called
vascular permeability factor (VPF), is a recently de-
scribed cytokine believed to be important in the patho-
genesis of diabetic nephropathy and retinopathy. Most
VEGF expression in the kidney is confined to the glo-
merular epithelial [53] and mesangial [54] cells. Ang II
stimulates the release of VEGF from human vascular tis-
sues [55] and mesangial cells [56]. Moreover, both Ang II
and mechanical stretch interact to increase VEGF pro-
S-96 Leehey et al: Angiotensin in diabetic nephropathy
Fig. 2. Collagenase activity from mesangial cells incubated in (A) 5
mmol/L glucose (NG) or 30 mmol/L glucose (HG) in the presence or
absence of losartan (L). Collagenase activity was measured in cell media
using fluorescein-labeled gelatin as substrate. Data are the means ⫾
SEM of 9 experiments. The asterisk denotes statistical significance (P ⬍
0.05) compared to NG and HG ⫹ L. (From [37]). (B) Collagenase
activity from mesangial cells incubated in either 5 mmol/L glucose (NG)
alone or in the presence of 10⫺6 M Ang II (NG ⫹ Ang II) or 10⫺6 M
Ang II plus 10⫺4 M losartan (NG ⫹ Ang II ⫹ L). Data shown are the
means ⫾ SEM of 8 experiments. The asterisk denotes statistical signifi-
cance (P ⬍ 0.05) compared to NG and NG ⫹ Ang II ⫹ L. (From [37]).
duction by mesangial cells [57]. In one study, glucose
was demonstrated to increase VPF mRNA expression
and protein secretion in vascular cells [58]. Further stud-
Fig. 3. Total TGF-␤1 secretion in media from mesangial cells incubated
in 5 mmol/L glucose (NG), 30 mmol/L glucose (HG), and HG ⫹ 10⫺4 M
losartan (HG ⫹ L) for 5 days. TGF-␤1 was measured by ELISA.
Incubation in high-glucose media produced a significant (P ⬍ 0.05)
increase in total TGF-␤1 that was blocked by losartan. (From [37]).
ies are needed to examine the effects of VEGF on matrix
accumulation in diabetes [59].
Interaction of the RAS with nitric oxide and
endothelin (ET) in diabetes
Endothelial cells are known to produce vasodilator sub-
stances such as nitric oxide (NO) and vasoconstrictor sub-
stances including endothelin-1 (ET-1). The NO system
appears to be upregulated in diabetic nephropathy [60],
where it serves as a functional antagonist of Ang II. NO
has renal antiproliferative effects and decreases matrix
protein synthesis [61, 62]. NO also inhibits the vasocon-
strictor actions of Ang II on preglomerular arterioles and
down-regulates the synthesis of ACE and AT1 receptors
[63]. Ang II activates nicotinamide adenine dinucleotide
(reduced form)/nicotinamide adenine dinucleotide phos-
phate (reduced form) oxidase and induces superoxide pro-
duction and hypertrophy by mesangial cells. The super-
oxide anion interacts with nitric oxide, thus reducing its
bioactivity, and acts as an intracellular signal leading to
cell growth [64]. Ang II is also known to be an important
stimulus for ET synthesis by endothelial cells and mesan-
gial cells [60] and ACE inhibitors and ARBs reduce ET.
The role of these endothelial substances and their rela-
tionship to Ang II in diabetic nephropathy require fur-
ther investigation.
Reprint requests to David J. Leehey, M.D., Hines Veterans Affairs
Hospital (111-L), Hines, Illinois, 60141 USA.
E-mail: david.leehey@med.va.gov
 Leehey et al: Angiotensin in diabetic nephropathy
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