Am J Physiol Renal Physiol 300: F301–F310, 2011.
First published December 8, 2010; doi:10.1152/ajprenal.00607.2010.
Review
Recent insights into diabetic renal injury from the db/db mouse model
of type 2 diabetic nephropathy
G. H. Tesch1,2 and A. K. H. Lim1,2
Department of 1Nephrology and 2Monash University Department of Medicine, Monash Medical Centre, Clayton, Victoria,
Australia
Submitted 18 October 2010; accepted in final form 1 December 2010
kidney; gene knockout; uninephrectomy; therapy
THE DB/DB MOUSE IS THE MOST widely used mouse model of type
2 diabetes. First described in 1966 (28), the db gene encodes a
G-to-T point mutation of the leptin receptor, which is transmitted in an autosomal recessive fashion. This leptin receptor
mutation (LepRdb/db) results in abnormal splicing and a defective receptor for the adipocyte-derived hormone leptin (6, 37).
This defect affects hypothalamic responses, leading to the
development of hyperphagia, obesity, hyperlipidemia, hyperinsulinemia, insulin resistance, and diabetes. Following the
onset of overt diabetes, db/db mice develop progressive kidney
disease, which has similarities to human diabetic nephropathy.
Consequently, db/db mice are now regularly used to examine
the mechanisms of renal injury which result from type 2
diabetes. Therapies which reduce obesity or hyperglycemia in
db/db mice also delay or prevent the onset of diabetic kidney
disease; however, such treatments are not discussed in the
current review.
The LepRdb/db mutant was originally recognized in the
C57BLKS/J strain but has been backcrossed onto both the
Address for reprint requests and other correspondence: G. Tesch. Dept. of
Nephrology, Monash Medical Centre, 246 Clayton Rd., Clayton, Victoria
3168, Australia (e-mail: Greg.Tesch@monash.edu.au).
http://www.ajprenal.org
C57BL/6J strain (27) and the FVB/NJ strain (12). In each of
these strains, diabetes is more severe in males compared with
females. Male C57BLKS/J db/db mice rapidly develop hyperglycemia (⬎16 mmol/l at 6 –10 wk), albuminuria (10 –12 wk),
and declining renal function (15–18 wk) (13, 39), but their islet
cells are sensitive to toxic effects of hyperglycemia, and these
mice become dependent on insulin administration to control
increasing blood glucose levels and allow survival. In comparison, the islets of the FVB/NJ db/db strain are more resistant to
hyperglycemia-induced injury and their insulin production is
better preserved in the presence of diabetes, which permits
longer survival without intervention. FVB/NJ db/db mice develop hyperglycemia at about 4 wk of age, which is maintained
at a similar level until at least 26 wk of age when mice develop
albuminuria and significant renal histological lesions (11, 12).
In contrast, the C57BL/6J db/db strain has a lower incidence of
diabetes with ⬃50% of males and 12% of females having
significant hyperglycemia (⬎16 mmol/l) at 32 wk of age (8).
C57BL/6J db/db males which develop diabetes have significant
albuminuria, renal histological damage, and reduced renal
function at 32 wk of age.
The rapid development of hyperglycemia and diabetic renal
injury in C57BLKS/J db/db mice has resulted in this strain
becoming the preferred db/db model for short-term interven-
0363-6127/11 Copyright © 2011 the American Physiological Society
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Tesch GH, Lim AK. Recent insights into diabetic renal injury from the db/db mouse
model of type 2 diabetic nephropathy. Am J Physiol Renal Physiol 300: F301–F310, 2011.
First published December 8, 2010; doi:10.1152/ajprenal.00607.2010.—The db/db mouse is
the most widely used animal model of type 2 diabetic nephropathy. Recent studies have
utilized genetic backcrossing with transgenic mouse strains to create novel db/db strains
that either lack or overexpress specific genes. These novel strains [ICAM-1⫺/⫺,
CCL2⫺/⫺, MKK3⫺/⫺, osteopontin⫺/⫺, plasminogen activator inhibitor-1 (PAI1)⫺/⫺, endothelial nitric oxide synthase⫺/⫺, SOD-Tg, rCAT-Tg] have provided
valuable insights into the molecular mechanisms which promote diabetic renal injury.
In addition, surgical removal of one kidney has been shown to accelerate injury in the
remaining kidney of diabetic db/db mice. A number of novel therapeutic agents have
also been tested in db/db mice, including inhibitors of inflammation (chemokine
receptor antagonists, anti-CCL2 RNA aptamer, anti-c-fms antibody); oxidative stress
(oxykine, biliverdin); the renin-angiotensin-aldosterone system (aliskiren, angiotensinconverting enzyme inhibitors, angiotensin receptor blockers, eplerenone); advanced
glycation end products (AGE; pyridoxamine, alagebrium, soluble AGE receptor);
angiogenesis (NM-3, anti-CXCL12 RNA aptamer, soluble Flt-1); lipid accumulation
(statins, farnesoid X receptor agonists, Omacor); intracellular signaling pathways
(PKC-␤ or JNK inhibitors); and fibrosis [transforming growth factor (TGF)-␤ antibody,
TGF-␤R kinase inhibitor, soluble betaglycan, SMP-534, CTGF-antisense oligonucleotide, mutant PAI-1, pirfenidone], which have identified potential therapeutic targets for
clinical translation. This review summarizes the advances in knowledge gained from
studies in genetically modified db/db mice and treatment of db/db mice with novel
therapeutic agents.
Review
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DIABETIC RENAL INJURY IN db/db MICE
tion studies. In contrast, the C57BL/6J db/db strain is more
commonly used for studies examining the effects of singlegene deficiencies or gene overexpression on the development
of diabetic renal injury, despite the slow rate of disease progression in this model. This is mainly due to the large range of
genetic modifications available on the C57BL/6J background
and the ease with which these modified strains can be backcrossed onto C57BL/6J db/db mice.
Acceleration of Nephropathy in db/db Mice by
Uninephrectomy
AJP-Renal Physiol • VOL
Studies of Gene Deficiencies in db/db Mice
Deficiency of ICAM-1. ICAM-1 plays a major role in recruiting leukocytes from the circulation into the kidney.
C57BL/6J db/db mice with genetic deficiency of ICAM-1
(ICAM-1⫺/⫺ db/db) were found to have an 85% reduction in
albuminuria at 32 wk of age compared with equally diabetic
wild-type controls (ICAM-1⫹/⫹ db/db mice) (10). This protection from renal injury was associated with reductions in glomerular leukocytes (63%2) and interstitial leukocytes
(83%2) and marked attenuation of glomerular and tubular
damage and kidney matrix accumulation (10). This was the
first study to indicate a functional role for leukocytes in the
development of diabetic renal injury in db/db mice.
Deficiency of MCP-1/Ccl2. Macrophages are the major leukocytes present in diabetic kidneys. Monocyte chemoattractant
protein (MCP)-1, encoded by the gene Ccl2, is a major chemokine for monocyte/macrophages. C57BL/6J db/db mice
deficient in Ccl2 have a reduced number of CD68⫹ macrophages in glomeruli (48%2) and the interstitium (78%2) at
32 wk of age compared with wild type db/db mice with
equivalent diabetes (9). In addition, diabetic Ccl2⫺/⫺ db/db
mice have reduced levels of albuminuria (62%2) and plasma
creatinine (65%2), which are associated with protection from
glomerular and tubular pathology and interstitial accumulation
of myofibroblasts and extracellular matrix. These findings
demonstrate a role for macrophage-mediated injury in the
diabetic kidneys of db/db mice.
Deficiency of osteopontin. Osteopontin (OPN), also known
as secreted phosphoprotein 1 (Spp1), is complex glycoprotein
which can promote cell adhesion and migration and has some
profibrotic properties. Its role in diabetic nephropathy has
recently been examined in OPN⫺/⫺ mice (129 ⫻ Black Swiss)
which were crossed onto the C57BLKS/J db/⫹ strain to produce OPN⫺/⫺ db/db mice (44). These OPN⫺/⫺ db/db mice
were found to have reduced albuminuria (at least 50%2) at
2– 4 mo of age and decreased glomerular levels of collagen IV,
fibronectin and TGF-␤ compared with equally diabetic
OPN⫹/⫹ db/db littermates. This study indicates that OPN
promotes diabetic renal injury in db/db mice; however, the
mechanisms responsible for this are still unclear and may
involve effects on leukocyte accumulation and activation or
damage to the glomerular filtration barrier induced by alterations to the glomerular structure.
Deficiency of MKK3. Elements of the diabetic milieu are
known to induce proinflammatory and profibrotic responses in
kidney cells via activation of the p38 MAPK signaling pathway. This pathway is activated in the kidneys of diabetic
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Use of the db/db mouse as a research tool is sometimes
limited by the rate of disease development and the severity of
renal function impairment and kidney lesions. To accelerate
diabetic renal injury in the db/db mouse, some researchers have
developed a model in which one kidney is removed (uninephrectomized) in the early stages of diabetes, which puts a
greater workload and stress on the remaining diabetic kidney.
The effects of uninephrectomy on glomerular injury were
first reported in the C57BLKS/J db/db strain in 1980 (4). In this
study, uninephrectomy was performed at 16 wk of age and
db/db mice were analyzed at 6 mo of age. Compared with
nonoperated db/db mice, the uninephrectomized db/db mice
had a 70% increase in mesangial thickening based on semiquantitative analysis of periodic acid-Schiff (PAS) staining. In
subsequent studies, uninephrectomy of db/db mice has been
performed at a much earlier age (6 –10 wk) to obtain more
profound renal effects at 5– 6 mo of age. C57BLKS/J db/db
mice which underwent uninephrectomy at 6 wk of age exhibit
exacerbation of renal function impairment and histological
damage at 6 mo of age compared with sham-operated db/db
controls. These uninephrectomized db/db mice have a 65%
increase in their albumin/creatinine ratio at 2 mo of age, which
progresses to a 240% increase at 5– 6 mo (46, 47). They also
exhibit a further 55– 60% reduction in glomerular filtration rate
(GFR) at 6 mo compared with their db/db controls (23, 45). In
terms of glomerulosclerosis, reports have indicated that uninephrectomy in C57BLKS/J db/db mice increases glomerular
deposits of PAS (40 –50%) and fibronectin (250%) and the
number of glomeruli with advanced sclerotic lesions (23,
45– 47). In contrast, there is currently a lack of data which
directly examine interstitial fibrosis in this model, although
some reports have indicated that uninephrectomy increases the
interstitial volume (50 –75%) and the number of tubules with
atrophy or dilatation (250 –300%) in these mice, suggesting an
exacerbation of tubulointerstitial damage (45– 47). In addition,
several studies have demonstrated that uninephrectomy promotes the recruitment of macrophages into glomeruli (200 –
280%) and the interstitium (70 –220%), which correlates with
a rise in kidney Ccl2 mRNA levels (200 –350%), demonstrating increased inflammation (45– 47, 53).
A micropuncture study performed in the C57BL/6J db/db
strain has shown that 3 wk of uninephrectomy increases whole
kidney GFR and single-nephron GFR compared with shamoperated db/db controls, while maintaining systolic blood pressure, proximal tubule reabsorptive rates, and an intact TGF
system (38). However, the increase in whole kidney GFR was
not significant after correction for the increase in kidney mass
in uninephrectomized mice. Further examination after 20 wk of
uninephrectomy showed that mesangial matrix expansion oc-
curs in the uninephrectomized C57BL/6J db/db mice compared
with sham-operated db/db controls.
Recent characterization of the accelerated nephropathy in
uninephrectomized db/db mice has resulted in growing use of
this model for studies examining the effects of pharmacological inhibitors on the development of diabetic renal injury (26,
45, 59) One advantage of this model is that it allows pharmacological responses to be determined in db/db mice in a shorter
time frame. However, when choosing this model, researchers
need to consider the technical expertise required, the surgical
mortality rate (⬃5%), and the limited amount of kidney tissue
obtained.
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DIABETIC RENAL INJURY IN db/db MICE
AJP-Renal Physiol • VOL
which is associated with fourfold higher insulin levels and
increased islet hypertrophy (41). One study also found a
twofold increase in plasma cholesterol level but no obvious
difference in aortic atherosclerosis at 16 wk (41).
In terms of renal injury, eNOS⫺/⫺ db/db mice have increased
albuminuria at 16 (430%1) and 26 wk (570%1) and reduced
renal function at 26 wk (inulin clearance 55%2, plasma
creatinine 48%1) compared with eNOS⫹/⫹ db/db mice. These
mice also showed increases in glomerular hypertrophy, glomerular matrix deposition, glomerular and tubular injury, and
kidney macrophage accumulation, which was evident at both
16 and 26 wk (41, 63). Furthermore, characteristic features of
human diabetic nephropathy, including arteriolar hyalinosis
and nodular glomerulosclerosis, can be regularly seen in
eNOS⫺/⫺ db/db, but are not normally detected in wild-type
db/db mice; however, interstitial fibrosis remains relatively
mild in db/db mice lacking eNOS. These findings indicate that
eNOS⫺/⫺ db/db mice represent an accelerated model of type 2
diabetic nephropathy which may prove useful for evaluating
novel therapies.
Effects of Gene Overexpression in db/db Mice
Recent studies of gene overexpression in db/db mice have
focused on increasing the production of antioxidant enzymes as
a means of reducing the pathological effects of oxidative stress
in diabetic kidneys. Hyperglycemia is known to induce reactive oxygen species (ROS) in kidney cells, which can directly
damage DNA and protein, or function as signaling amplifiers
to activate pathways of cellular stress.
Overexpression of SOD-1. C57BL/6J mice expressing the
human Cu/Zn-SOD-1 transgene driven by the human SOD-1
promoter were crossed with C57BL/6J db/⫹ mice to create the
SOD-1⫹/⫺ db/db genotype (SOD-Tg-db/db) (19). These transgenic mice have a greater than twofold increase in cortical
SOD-1 levels compared with wild-type db/db mice, which is
mainly localized to the glomerular endothelial and tubular
epithelial cells. The SOD-Tg-db/db mice were completely
protected from the threefold increase in albuminuria seen in
wild-type db/db mice at 5 mo of age, despite their hyperfiltration (increased inulin clearance). Furthermore, SOD-Tg-db/db
mice had reduced glomerular levels of mesangial matrix,
TGF-␤, and nitrotyrosine compared with control db/db mice.
The investigators also examined the effects of treatment with
N␻-nitro-L-arginine methyl ester (L-NAME), an inhibitor of
NOS on diabetic mice. Inulin clearance and urinary cGMP
were suppressed by L-NAME in db/db-SOD-Tg but not in
db/db mice, suggesting that the greater hyperfiltration in db/
db-SOD-Tg mice was due to enhanced renal nitric oxide (NO)
bioactivity. When cGMP responses to NO stimulation were
examined ex vivo, glomeruli from db/db mice demonstrated
greater quenching of NO compared with db/db-SOD-Tg mice.
These findings demonstrate that superoxide mediates biochemical, functional, and structural changes in the diabetic kidneys
of db/db mice, which appear, in part, to involve interactions
with NO.
Overexpression of catalase. Apoptosis is seen in the proximal tubular cells of diabetic kidneys, which may contribute to
the development of tubular atrophy during diabetic nephropathy. One potential cause of this apoptosis is the generation of
ROS in tubular cells by hyperglycemia. A recent study has
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patients and diabetic db/db mice (1). Because mice deficient in
p38␣ die in utero, the functional role of p38 MAPK cannot be
examined directly by genetic deletion in db/db mice. However,
mice genetically deficient in MKK3 or MKK6, the upstream
kinases of p38 MAPK, are viable. C57BL/6J db/db mice
deficient in mitogen activated kinase kinase-3 (Mkk3⫺/⫺ db/
db) have reduced kidney activation of p38 MAPK and develop
less renal injury compared with equally diabetic wild-type
controls (Mkk3⫹/⫹ db/db) (40). At 32 wk of age, diabetic
Mkk3⫺/⫺ db/db mice have decreased levels of albuminuria
(70%2) and are protected from the loss of renal function
observed in diabetic db/db controls. In terms of kidney structure, diabetic Mkk3⫺/⫺ db/db mice have reductions in podocyte
loss, tubular damage, and glomerular and interstitial accumulation of myofibroblasts and extracellular matrix. In addition,
these mice have lower numbers of interstitial macrophages,
which is associated with reduced kidney gene expression of
Ccl2. These findings support a role for MKK3-p38MAPK
signaling in renal injury, inflammation, and fibrosis in the
diabetic kidneys of db/db mice.
Deficiency of plasminogen activator inhibitor-1. Plasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor
which is thought to function as a profibrotic mediator in
diabetic nephropathy. PAI-1 inhibits tissue- and urokinase-type
plasminogen activators. The effects of PAI-1 on diabetic renal
fibrosis have been evaluated in PAI-1⫺/⫺ mice (B6/129) which
were crossed onto the C57BLKS/J db/⫹ strain to produce
PAI-1⫺/⫺ db/db mice (16). The kidneys of PAI-1⫺/⫺ db/db
mice showed 60% higher levels of urokinase activity compared
with PAI-1⫹/⫹ db/db littermate controls, indicating that higher
PAI-1 levels in db/db mice suppressed urokinase activity. At 8
mo of age, PAI-1⫺/⫺ db/db mice showed a trend toward
reduced albuminuria; however, this was not statistically significant due to high variability. In addition, PAI-1 deficiency did
not affect renal hypertrophy or blood urea nitrogen levels in
db/db mice. Diabetic PAI-1⫺/⫺ db/db mice also had a similar
glomerular matrix fraction and total kidney collagen content to
wild-type db/db controls. The investigators highlighted several
concerning issues with the study. First, only females were
studied because the PA1–1⫺/⫺ db/db phenotype was associated
with a greater risk of premature death and runtiness in males.
PAI-1⫺/⫺ db/db mice also had 15–25% lower blood glucose
levels and a trend toward lower lipid levels than PAI-1⫹/⫹
db/db mice, suggestive of other metabolic consequences of
PAI-1 deficiency.
Deficiency of endothelial nitric oxide synthase. Endothelial
dysfunction is implicated in the pathogenesis of diabetic nephropathy. Endothelial nitric oxide synthase (eNOS) modulates functions such as permeability, vascular tone, blood flow,
and hyperfiltration. Mice with a genetic deficiency of eNOS are
inherently hypertensive and develop slowly progressing renal
damage (18). To develop an accelerated model of type 2
diabetic nephropathy, eNOS⫺/⫺ mice have been backcrossed
onto the C57BLKS/J strain and then crossed with C57BLKS/J
db/⫹ mice to produce eNOS⫺/⫺ db/db mice (63). The
eNOS⫺/⫺ db/db mouse has some phenotypic differences compared with eNOS⫹/⫹ db/db mice. First, these mice develop
moderate systolic hypertension, which is ⬃40% higher than
eNOS⫹/⫹ db/db mice, but is not different to eNOS⫺/⫺ mice
(41, 63). Second, they have lower fasting blood glucose levels
(25%2) at 16 wk of age compared with eNOS⫹/⫹ db/db mice,
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Therapeutic Agents Used in db/db Mice
Inhibitors of inflammation. Studies in db/db mice deficient in
ICAM-1 or MCP-1 have demonstrated a critical role for
macrophage-mediated inflammation in the development of
diabetic renal injury. In response to these findings, researchers
have developed novel therapeutic strategies for targeting macrophages and have evaluated their effectiveness in db/db mice.
Administration of BL5923, a CCR1 antagonist, to uninephrectomized C57BLKS/J db/db mice between 5 and 6 mo of
age was found to reduce white blood cell counts and interstitial
macrophage accumulation, as well as the development of
tubular damage and interstitial fibrosis (46). However, treatment with BL5923 had no effect on albuminuria or glomerular
pathology. In another study, uninephrectomized C57BLKS/J
db/db mice receiving mNOX-E36 –3=PEG, an anti-CCL2 Lenantiomeric RNA aptamer, between 4 and 6 mo of age, were
found to have normal white blood cell counts but showed a
marked reduction in glomerular and interstitial macrophages.
This RNA aptamer provided protection from declining GFR,
glomerular and tubulointerstitial damage, and renal fibrosis
(45). Macrophages have also been targeted in db/db mice using
a neutralizing antibody to the macrophage CSF-1 receptor
(c-fms). Treatment of C57BLKS/J db/db mice with a c-fms
antibody (AFS98) from 12–18 wk of age had no effect on
white blood cell counts but reduced both glomerular and
interstitial macrophages (39). AFS98 also protected db/db mice
from glomerular hyperfiltration, tubular injury, and mild interstitial fibrosis; however, established albuminuria and glomerular pathology were unaltered by this therapy.
AJP-Renal Physiol • VOL
Inhibitors of oxidative stress. Novel antioxidants have been
tested in db/db mice to determine their potential for reducing
oxidative stress and injury in diabetic kidneys. Oxykine is a
melon extract rich in vegetal SOD which is protected from
digestive degradation by a polymeric film of gliadin. Oral
administration of oxykine to C57BLKS/J db/db mice between
6 and 18 wk of age has no effect on the development of
diabetes but reduces urine levels of albumin and 8-hydroxydeoxyguanosine, and the mesangial matrix fraction (43).
Biliverdin is a precursor of bilirubin, which is recognized as an
endogenous antioxidant with a highly efficient radical-scavenging effect. C57BLKS/J db/db mice maintained on a diet
supplemented with biliverdin from week 8 to week 10 or 20
were found to be protected from renal oxidative stress and
albuminuria. Those mice treated for 12 wk also had reduced
accumulation of mesangial matrix (22).
Inhibitors of the renin-angiotensin-aldosterone system. The
use of an ACE inhibitor (ACEi) or an angiotensin II receptor
blocker (ARB) to control hypertension is standard therapy for
patients with diabetic nephropathy. The renoprotective effects
of these therapies and other inhibitors of the renin-angiotensinaldosterone system (RAAS) pathway have been examined in
db/db mice.
Treatment of C57BLKS/J db/db mice with the ARB losartan
from 11–29 wk of age has been shown to partially reduce
albuminuria and lipid peroxidation, but not mesangial matrix
expansion (42). In another study, the effects of both an ACEi
(benazepril) and an ARB (valsartan) were examined in normotensive C57BL/6 db/db mice to identify whether blocking these
pathways had protective effects beyond inhibiting hypertension
(64). Treatment of C57BL/6 db/db mice with benazepril and
valsartan from 9 to 25 wk of age was found to reduce albuminuria, glomerular hypertrophy, and mesangial matrix accumulation. However, the same study found no effect when db/db
mice were treated with an ACEi (enalapril) alone.
Administration of a direct renin inhibitor, aliskiren, to
C57BLKS/J db/db mice between 6 and 12 wk of age was found
to reduce circulating levels of angiotensin II but had no effect
on blood pressure in these mice. Mice treated with aliskiren
had reduced levels of albuminuria, renal oxidative stress,
glomerular inflammation, mesangial matrix, and glomerular
TGF-␤1 and were protected from a loss of nephrin expression
(20). The level of this protection was increased further when
mice were treated with aliskiren in combination with valsartan,
which reduced blood pressure.
Eplerenone, a specific antagonist of the mineralocorticoid
receptor, has been used to examine whether aldosterone blockade can reduce diabetic renal injury. Administration of
eplerenone to C57BLKS/J db/db mice between 8 and 25 wk of
age had no effect on diabetes or systolic blood pressure, but
decreased albuminuria, glomerular hypertrophy, and mesangial
matrix accumulation (25). Eplerenone treatment also lowered
kidney mRNA levels of OPN, CD68, and TGF-␤1, suggesting
that aldosterone promotes inflammation and fibrosis in diabetic
kidneys.
Inhibitors of advanced glycation end products. Diabetes
induces the formation of advanced glycation end products
(AGEs), which can modify the function of proteins and stimulate pathological cellular responses via AGE receptors. Increasing levels of AGEs, and their deposition in diabetic
kidneys, correlate with the development of diabetic nephropa-
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examined whether overexpression of the antioxidant enzyme
catalase (CAT) in proximal tubular cells is capable of preventing tubular apoptosis and renal injury in diabetic db/db mice
(5). Transgenic C57BL/6J mice overexpressing rat catalase
(rCAT) in their renal proximal tubular cells were crossed with
the C57BL/6J db/⫹ strain to produce rCAT-Tg-db/db mice (5).
In these mice, the expression of the rCAT-HA transgene is
under the control of the kidney-specific androgen-regulated
(KAP2) promoter, which is stimulated by circulating endogenous testosterone in males or exogenously administered testosterone in females. At 20 wk of age, there was a marked
reduction in the ROS level (73%2) of renal proximal tubular
cells isolated from rCAT-Tg-db/db mice compared with those
isolated from wild-type db/db mice. The db/db and rCAT-Tgdb/db mice developed similar obesity and hyperglycemia during the analysis period (8 –20 wk). However, wild-type db/db
mice showed mild hypertension (20 –30 mmHg 1systolic
blood pressure) after 12–14 wk of age compared with the
rCAT-Tg-db/db or db/⫹ groups. Urine analysis showed that
albuminuria was elevated 10- to 20-fold in db/db mice at weeks
18 –20 compared with nondiabetic db/⫹ mice, but was reduced
in the rCAT-Tg-db/db by 36 – 42% in males and by 67– 83% in
females. Examination of the diabetic kidneys at 20 wk found
reduced tubular apoptosis and interstitial fibrosis in rCAT-Tgdb/db mice but no difference in glomerular hypertrophy compared with db/db mice. These findings indicate that intrarenal
ROS plays a role in the development of albuminuria, tubular
injury, and interstitial fibrosis in the diabetic kidneys of db/db
mice.
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DIABETIC RENAL INJURY IN db/db MICE
AJP-Renal Physiol • VOL
matrix, the glomerular capillary area, glomerular macrophages,
and nephrin mRNA expression. However, the impact of treatment on tubulointerstitial injury was not reported.
Chemokine (C-X-C motif) ligand 12 (CXCL12), also known
as stromal cell-derived factor (SDF)-1, is known to induce
angiogenesis, support vascular integrity similar to VEGF, and
promote cell migration and survival. Administration of NOXA12, an anti-CXCL12 L-enantiomeric RNA aptamer, to uninephrectomized C57BLKS/J db/db mice between 4 and 6 mo
of age was found to reduce albuminuria, glomerulosclerosis,
and podocyte loss, but had no effect on macrophage accumulation or cell proliferation within glomeruli (53).
Soluble Flt-1 (sFlt-1) is a variant form of the VEGF receptor
1, which lacks the transmembrane and intracellular domain.
Binding of sFlt-1 to VEGF neutralizes its action on endogenous VEGF receptors. One study has used an adenoviralassociated virus as a vector to introduce sFlt-1 into 8-wk-old
male C57BLKS/J db/db mice, which resulted in an attenuation
of diabetes-induced elevation of phospho-Flt-1 after 8 wk of
treatment (34). Low-dose gene therapy did not alter blood
pressure, body weight, or hyperglycemia. However, albuminuria was attenuated by ⬃50% in sFlt-1-treated db/db mice,
which was associated with protection from podocyte injury and
depletion. In contrast, overexpression of sFlt-1 did not alter
glomerular matrix accumulation and exacerbated tubulointerstitial injury, which included a loss of peritubular capillaries.
This was associated with a reduction in tubular but not glomerular VEGF expression. These findings demonstrate the
adverse effects of systemic blockade of VEGF and emphasize
the difficulty in targeting the pathological effects of VEGF in
diabetic kidneys.
Inhibitors of lipid accumulation. Statins are widely used
cholesterol-lowering agents but have also been reported to
have anti-inflammatory and antioxidative actions. Administration of pitavastatin to C57BLKS/J db/db mice between 12 and
14 wk of age was found to reduce albuminuria and kidney
oxidative stress, which included a decrease in kidney NOX4
production (21). This report also demonstrated that long-term
treatment from 12 to 24 wk decreased mesangial matrix and
kidney mRNA levels of TGF-␤1 and fibronectin. In a similar
study, treatment of C57BLKS/J db/db mice with simvastatin
from 8 to 24 wk of age reduced albuminuria and mesangial
matrix accumulation in association with attenuation of kidney
activity of the small GTPase protein RhoA (33). These findings
suggest that statins protect against diabetic renal injury, in part,
by inhibiting renal oxidative stress and RhoA activity.
The farnesoid X receptor (FXR) is a member of the nuclear
hormone receptor superfamily, which is highly expressed in
the kidney and liver and is known to regulate lipid metabolism
by inhibiting the sterol regulatory element binding protein-1
(SREBP-1). Levels of SREBP-1 are increased in diabetic
kidneys and correlate with the progression of renal injury.
Treatment of 12-wk-old C57BLKS/J db/db mice with an FXR
agonist (GW4064) for 1 wk had no impact on hyperglycemia
but reduced plasma cholesterol and kidney mRNA expression
of SREBP-1, Nox2, proinflammatory cytokines, and profibrotic molecules (31). In a follow-up study, C57BL/6 db/db
mice were treated with another FXR agonist, cholic acid,
between 12 and 24 wk of age, which reduced albuminuria and
mesangial matrix accumulation. However, the control and
treated C57BL/6 db/db mice were not overtly diabetic (31).
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thy. Several recent studies have tested the effectiveness of
novel inhibitors of AGE formation for preventing the progression of diabetic renal injury in db/db mice.
Cohen et al. (15) have examined a variety of AGE inhibitors
in db/db mice. Their early studies showed that weekly administration of an anti-AGE-albumin monoclonal antibody A717
was able to decrease circulating levels of AGE-albumin in
C57BLKS/J db/db mice, which reduced albuminuria, mesangial matrix and cortical gene expression of matrix components
(15). Subsequent studies by this group have examined a number of small-molecule inhibitors of AGE formation as potential
therapies, with the most recent being designated 23CPPA (14).
Treatment of C57BLKS/J db/db mice with 23CPPA from 9 to
18 wk of age had no effect on diabetes but reduced kidney
hypertrophy, urine excretion of albumin and collagen IV, and
cortical levels of VEGF and VEGFR1. In addition, 23CPPA
protected these db/db mice from declining renal function and
loss of nephrin protein in the renal cortex.
Pyridoxamine, another inhibitor of AGE formation, has been
shown to reduce albuminuria, glomerular hypertrophy, and
mesangial matrix in female C57BL/6J db/db mice following
treatment between 17 and 25 wk of age (64). Furthermore, this
study also showed that the combined administration of an ACE
inhibitor (enalapril) and pyridoxamine from 16 –32 wk provided added protection against renal injury and mortality compared with the use of enalapril alone.
Alagebrium (ALT-711), an AGE cross-link breaker, has
been used to treat female C57BLKS/J db/db mice, which
develop diabetic nephropathy more slowly than their male
littermates (48). In this study, 12 wk of alagebrium treatment
commencing at either 3, 7, or 12 mo of age reduced serum
AGE levels and the progression of albuminuria, glomerular
hypertrophy, and mesangial matrix accumulation. In addition,
kidney AGE deposition and glomerular basement width were
diminished following treatment which began at 7 and 12 mo.
These results suggest that alagebrium may prevent the progression of early and advanced diabetic nephropathy.
Diabetic kidneys have elevated expression of the receptor for
AGE (RAGE) as well as an increased number of ligands for this
receptor, including AGEs and S100/calgranulin. A soluble form
of RAGE (sRAGE) is known to block AGEs and S100/calgranulin from binding to RAGE on cells and inducing pathological
responses. Administration of sRAGE to C57BLKS/J db/db mice
between 8 and 13 wk of age reduced kidney levels of RAGE,
S100/calgranulin, VEGF, VCAM-1, and prevented glomerular
macrophage infiltration (62). In comparison, longer term administration of sRAGE between 8 and 27 wk of age protected
diabetic db/db mice from declining renal function and also
decreased albuminuria, glomerular hypertrophy, mesangial
matrix accumulation, glomerular basement membrane thickening, and expression of VEGF and TGF-␤. These findings
indicate that RAGE activation plays an important role in the
development of diabetic nephropathy.
Inhibitors of angiogenesis. The progression of diabetic nephropathy is associated with an increase of angiogenic factors
such as VEGF and angiopoietin (Ang)-2, which synergize to
promote angiogenesis. Daily intraperitoneal injection of the
antiangiogenic compound NM-3 to C57BLKS/J db/db mice
between 8 and 16 wk of age lowered kidney the levels of
VEGF-A and Ang-2 (29). This treatment reduced albuminuria,
glomerular hyperfiltration, glomerular hypertrophy, mesangial
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TGF-␤1/p38 MAPK activity, SMP-534, to C57BLKS/J db/db
mice between 9 and 25 wk of age (58). SMP-534 decreased
mesangial matrix expansion and urine excretion of albumin
and collagen IV. These findings indicate that TGF-␤1 plays a
critical role in diabetic glomerulosclerosis in db/db mice;
however, its involvement in the development of diabetic albuminuria remains controversial.
Connective tissue growth factor (CTGF) is another profibrotic cytokine which promotes matrix accumulation in diabetic kidneys. Antisense oligonucleotides targeting CTGF have
been given as a therapy to C57BLKS/J db/db mice between 12
and 20 wk of age and were found to reduce CTGF levels in the
renal cortex and urine (24). These mice were protected from
increases in albuminuria, renal dysfunction, mesangial matrix
accumulation, and kidney mRNA levels of collagen (I and IV)
and PAI-1.
PAI-1 production is induced by TGF-␤1 and CTGF and
contributes to matrix accumulation in diabetic kidneys by
reducing plasmin activity and matrix degradation. A human
mutant form of PAI-1 (designated PAI-1R) has recently been
shown to compete with PAI-1 for vitronectin binding. Administration of PAI-1R to uninephrectomized C57BLKS/J db/db
mice between 20 and 22 wk of age increased kidney plasmin
activity but reduced TGF-␤1 protein, glomerular matrix, and
mRNA and protein levels of collagen (I and IV) and fibronectin
in the renal cortex (26).
In addition, PAI-1R therapy prevented an increase in albuminuria during the treatment period. This finding supports an
important role for endogenous PAI-1 activity in the development of diabetic glomerulosclerosis in db/db mice.
Pirfenidone is a potent antifibrotic compound, whose mechanisms of action are still being defined. Treatment of
C57BLKS/J db/db mice with pirfenidone from 17 to 21 wk of
age reduced mesangial matrix accumulation and kidney cortex
mRNA levels of collagen (I and IV) and fibronectin but did not
decrease albuminuria (51). Subsequent analysis identified 21
proteins that are unique to pirfenidone-treated db/db kidneys
and suggest that the antifibrotic effects of pirfenidone may
include regulation of RNA processing.
Discussion
The growing incidence of type 2 diabetic nephropathy has
driven a major effort to develop a greater understanding of the
mechanisms underlying this disease and to explore novel
intervention strategies as potential therapies. This has led to an
increase in use of the db/db mouse model as a research tool.
Recent advances in genetic manipulation and pharmacology
have been applied to the db/db model to determine the importance of specific mechanisms in type 2 diabetic nephropathy
and to identify therapeutic targets. In addition, the development
of accelerated models of diabetic renal injury in db/db mice,
using uninephrectomy or eNOS deficiency, provides increased
flexibility when one is choosing a db/db model for exploring
new mechanisms or therapies.
Studies in db/db mice have identified major mechanistic
pathways of diabetic renal injury (Fig. 1), which can be
suppressed by therapies which target key components of these
pathways (see Table 2). These pathomechanisms also appear to
be important in the development of rodent models of type 1
diabetic nephropathy. Lean mice which are genetically defi-
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These findings indicate that FXR agonists may suppress the
development of diabetic nephropathy due to systemic and renal
effects on lipid metabolism and by decreasing oxidative stress,
inflammation, and fibrosis in the kidney.
Investigators have also used Omacor, a solution containing n-3
polyunsaturated fatty acids, to suppress SREBP-1 activity in
diabetic kidneys (7). Administration of Omacor to C57BLKS/J
db/db mice between 8 and 10 wk of age did not alter diabetes
but lowered kidney levels of SREBP-1 and reduced serum
triglyceride, albuminuria, glomerular hyperfiltration, podocyte
loss, and mesangial matrix. However, Omacor treatment also
reduced food and water intake, suggesting that the observed
protection involves regulation of metabolism as well as lipid
signaling.
Inhibitors of intracellular signaling pathways. Diabetes regulates intracellular signaling responses, including those mediated by PKC-␤ and JNK. Both of these kinases are activated in
diabetic kidneys, which has prompted studies to block their
action in db/db mice.
Treatment of C57BLKS/J db/db mice with a PKC-␤ inhibitor (LY333531) from 9 to 25 wk of age reduced albuminuria,
glomerular TGF-␤ production, and mesangial matrix accumulation (35). In comparison, when C57BLKS/J db/db mice were
treated with a cell-permeable TAT-JNK inhibitor peptide from
8 to 18 wk of age, their insulin sensitivity improved and
hyperglycemia declined compared with db/db controls (30).
However, mice treated with the JNK inhibitor had increased
albuminuria, reduced nephrin expression, and no effect on
mesangial expansion. This finding indicates that therapies
which reduce the severity of diabetes do not necessarily provide renal protection.
Inhibitors of fibrosis. TGF-␤1 is known to be a major
inducer of profibrotic responses in diabetic kidneys. An earlier
study has shown that administration of a neutralizing monoclonal TGF-␤1 antibody to C57BLKS/J db/db mice between 8
and 16 wk of age decreases plasma TGF-␤1, mesangial matrix
expansion, and kidney mRNA levels of collagen IV and
fibronectin (65). In addition, this therapy prevented a loss of
renal function but had no effect on the elevated albuminuria in
db/db mice. More recently, investigators have used an inhibitor
of TGF-␤ receptor kinase activity, GW788388, to treat
C57BLKS/J db/db mice at 6 mo of age for 5 wk (49). This
therapy reduced glomerular collagen staining and kidney
mRNA levels of PAI-1 and collagen (I and III) but did not alter
albuminuria. A study has also identified that TGF-␤ activity
can be blocked in db/db mice using soluble betaglycan, which
binds to TGF-␤ and prevents activation of its receptors (32). In
this report, C57BLKS/J db/db mice were treated with soluble
betaglycan from 8 to 16 wk of age and were found to be
protected from albuminuria, declining renal function, and mesangial matrix expansion. Soluble betaglycan also reduced kidney mRNA levels of TGF-␤ isoforms, collagen (I and IV), and
fibronectin. Using a different strategy, investigators have
shown that administration of a naturally occurring inhibitor of
TGF-␤-induced SMAD signaling, N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SKD), to C57BLKS/J db/db mice between 10
and 18 wk of age reduces renal dysfunction, glomerular hypertrophy, mesangial matrix expansion, and overproduction of
extracellular matrix proteins (56). However, Ac-SKD did not
diminish albuminuria in these mice. In another approach,
researchers administered a low-molecular-weight inhibitor of
Review
DIABETIC RENAL INJURY IN db/db MICE
cient in ICAM-1, MCP-1, or PAI-1, or genetically overexpress
SOD, display protection against renal injury after induction of
diabetes with streptozotocin, which is similar to that seen in
obese db/db mice (17, 36, 60). In addition, therapeutic blockade of AGE formation (alagebrium), RAAS activation (losartan, eplerenone), or TGF-␤-induced fibrosis (TGF-␤ antibody)
can inhibit renal injury in the diabetic kidneys of db/db mice to
a similar extent as those of streptozotocin-treated rodents with
comparable diabetes (25, 52, 55, 61). This suggests that there
are common mechanisms of renal injury which are critical to
both type 1 and type 2 diabetic nephropathy. Furthermore,
inhibition of diabetic renal injury by blocking AGE formation
(pyridoxamine) or angiotensin II effects (losartan) is similarly
effective in obese Zucker fa/fa rats and db/db mice, which
demonstrates that the molecular mechanisms of renal injury are
alike in these models of type 2 diabetic nephropathy (2, 50).
Studies examining genetic modifications of db/db mice have
demonstrated a role for inflammation, oxidative stress, and p38
MAPK signaling in the development of type 2 diabetic nephropathy, which have been supported by intervention studies
with selective inhibitors (see Tables 1 and 2). In addition, the
use of pharmacological inhibitors in db/db mice has identified
additional molecular mechanisms that contribute to the progression of diabetic renal injury, including members of the
RAAS pathway (renin, angiotensin II, aldosterone), the lipid
signaling pathway (Fanesoid X receptor, SREBP-1), angiogenic factors (VEGF, CXCL12, angiopoietin-2), AGE, PKC-␤
signaling, and profibrotic molecules (TGF-␤1, CTGF, PAI-1).
Evidence provided from both genetic manipulation studies
and therapeutic interventions in db/db mice have demonstrated
a correlation between podocyte damage/loss and albuminuria
levels (7, 14, 20, 29, 34, 39, 40), suggesting that podocyte
injury is a critical factor in the early development of type 2
diabetic nephropathy. Furthermore, intervention studies in
db/db mice with established albuminuria indicate that many
strategies which effectively suppress mechanisms of renal
injury are unable to reduce albumin excretion below the pretreatment level, or sometimes not at all (39, 49, 51, 56, 65).
This emphasizes a difficulty in replacing podocytes which are
lost from diabetic glomeruli. It also indicates that intervention
therapies may provide significant renoprotection without reducing albuminuria, which suggests that albuminuria should
not be the only end point used for intervention studies.
The role of hypertension in the development of diabetic
renal injury in db/db mice has been controversial. Studies
employing tail-cuff measurements have produced mixed results, identifying increased, decreased, or no change in blood
pressure in db/db mice (5, 25, 35, 42). However, two reports
using radiotelemetric probes have recently shown that there is
a significant increase in mean arterial blood pressure (8 –16%)
at 14 –16 wk of age in C57BLKS/J db/db mice compared with
db/⫹ controls (54, 57). In contrast, radiotelemetry analysis has
not identified any evidence of hypertension in C57BL/6 db/db
Table 1. Summary of gene modifications in db/db mice with renal effects
Modified Gene (and Strain)
Intended Target
ICAM-1 KO (db/db BL6)
Leukocyte recruitment
MCP-1 KO (db/db BL6)
Macrophage recruitment and activation
OPN KO (db/db BKS)
MKK3 KO (db/db BL6)
Cell motility and activation
p38 MAPK signaling
PAI-1 KO (db/db BKS)
Matrix accumulation
eNOS KO (db/db BKS)
Endothelial function and vascular tone
SOD-Tg (db/db BL6)
Oxidative stress
rCAT-Tg (db/db BL6)
Oxidative stress in PTC
Effect on Diabetic Nephropathy
Reference No.
Reduces renal inflammation, albuminuria, glomerular
and tubular injury, and kidney fibrosis.
Reduces renal inflammation, albuminuria, glomerular
and tubular injury, and kidney fibrosis. Improves
GFR.
Reduces albuminuria and glomerular fibrosis.
Reduces podocyte loss, interstitial inflammation,
albuminuria, histological damage and kidney
fibrosis. Improves GFR.
Reduces hyperglycemia, cholesterol and glomerular
matrix fraction.
Induces hypertension. Exacerbates glomerular and
tubular damage, albuminuria, inflammation and
kidney fibrosis. Reduces GFR.
Reduces oxidative stress, albuminuria and
glomerular matrix. Increases GFR.
Reduces oxidative stress, hypertension, albuminuria,
tubular injury and interstitial fibrosis.
10
9
44
40
16
63
41
19
5
KO, knockout; OPN, osteopontin; PAI-1, plasminogen activator inhibitor-1; eNOS, endothelial nitric oxide synthase; Tg, transgenic; GFR, glomerular filtration
rate; PTC, proximal tubular cells.
AJP-Renal Physiol • VOL
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Fig. 1. Major mechanistic pathways of diabetic renal injury identified in db/db
mice. The development of diabetes in db/db mice results in the formation of
advanced glycation end products (AGEs), oxidative stress, and activation of
the renin-angiotensin-aldosterone system (RAAS) within the kidney, which
promotes progressive inflammation and fibrosis, leading to diabetic nephropathy and declining renal function. Intervention therapies described in Table 2
can inhibit each of these pathways in db/db mice.
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Table 2. Summary of recent intervention therapies examined in db/db mice
Mechanism and Compound
CCR1 antagonist
Anti-CCL2 RNA aptamer
c-fms Antibody
Vegetal SOD
Radical scavenger
ARB
ACEi
ACEi⫹ARB
Renin inhibitor
MR antagonist
Soluble AGE receptor
AGE inhibitor
AGE cross-link breaker
Inhibits
Albuminuria
Improves
GFR
Reduces Renal
Damage
Reference
No.
22–26wk
18–26wk
12–18wk
No
ND
No
ND
Yes
Yes
Yes
Yes
Yes
46
45
39
6–18wk
8–20wk
Yes
Yes
ND
ND
Yes
Yes
43
22
11–29wk
9–25wk
9–25wk
6–12wk
8–25wk
Yes
No
Yes
Yes
Yes
ND
ND
ND
ND
ND
No
No
Yes
Yes
Yes
42
64
64
20
25
Yes
Yes
Yes
Yes
ND
ND
Yes
Yes
Yes
62
64
48
Treatment Period, age
8–27wk
17–25wk
12–24, 30–42, 52–64 wk
Angiogenesis inhibitor
anti-CXCL12 RNA aptamer
Soluble VEGFR1
8–16wk
18–26wk
8–16wk
Yes
Yes
Yes
Yes
ND
No
Yes
Yes
No
29
53
34
Cholesterol lowering
Cholesterol lowering
FXR antagonist
SREBP-1 inhibitor
12–24wk
8–24wk
12–24wk
8–10wk
Yes
Yes
Yes
Yes
ND
ND
ND
Yes
Yes
Yes
Yes
Yes
21
33
31
7
9–25wk
8–18wk
Yes
No
ND
ND
Yes
No
35
30
8–16wk
26–31wk
8–16wk
10–18wk
9–25wk
12–20wk
20–22wk
17–12wk
No
No
Yes
No
Yes
Yes
Yes
No
Yes
ND
Yes
Yes
ND
Yes
ND
ND
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
65
49
32
56
58
24
26
51
PKC-␤ inhibitor
JNK inhibitor
TGF-␤1 antibody
TGF-␤R kinase inhibitor
Soluble TGF-␤R
SMAD inhibitor
TGF-␤/p38 MAPK inhibitor
CTGF gene transcription
Mutant PAI-1
Fibrosis inhibitor
GFR, glomerular filtration rate; AGE, advanced glycation end products; RAAS, renin-angiotensin-aldosterone system; ND, not determined.
mice (3). These findings indicate that it is feasible to evaluate
the effects of therapies on hypertension in db/db strains which
develop elevated blood pressure. However, the considerable
expense of radiotelemetry suggests that it would only be
sensible to use this analytic technique in db/db mice when the
effects of a therapy or genetic deficiency which is known to
have antihypertensive effects are being explored.
A limitation of recent studies of diabetic nephropathy in
db/db mice is that they are predominantly focused on glomerular damage and albuminuria as end points and rarely report
the development of tubulointerstitial injury. This is generally
due to the mild nature of the interstitial fibrosis in this model.
It is hoped that future development and characterization of
accelerated db/db models will enable the study of interstitial
fibrosis, which is featured in advanced diabetic nephropathy in
patients. The importance of this lies in the knowledge that the
interstitial fibrosis score is a strong predictor of end-stage renal
failure in biopsied diabetic patients.
In conclusion, the recent application of genetic modifications, uninephrectomy, and pharmacological inhibitors to
db/db mice has provided valuable knowledge about the pathomechanisms of diabetic nephropathy. Modifications of this
AJP-Renal Physiol • VOL
model are continuing to evolve, which may provide a greater
resemblance to human disease and be used to identify effective
new therapies.
GRANTS
G. H. Tesch is supported by a Career Development Award from the
National Health and Medical Research Council of Australia, Kidney Health
Australia, and the Australian and New Zealand Society of Nephrology.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
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