NEPHROLOGY 2007; 12, 261–266 doi:10.1111/j.1440-1797.2007.00796.

Methods in Renal Research

Rodent models of streptozotocin-induced diabetic nephropathy

GREG H TESCH1,2 and TERRI J ALLEN3

Departments of 1Nephrology and 2Medicine, Monash University, Monash Medical Centre, Clayton, and 3Einstein
JDRF Centre for Diabetic Complications, Baker Heart Research Institute, Melbourne, Victoria, Australia

SUMMARY: Streptozotocin-induced pancreatic injury is commonly used for creating rodent models of type 1
diabetes which develop renal injury with similarities to human diabetic nephropathy. This model can be estab-
lished in genetically modified rodents for investigating the role of molecular mechanisms and genetic susceptibility
in the development of diabetic nephropathy. In this report, the authors describe and compare the current protocols
being used to establish models of diabetic nephropathy in rat and mouse strains using streptozotocin. The
authors also list some of the histological criteria and biochemical measurements which are being used to validate
these models. In addition, our review explains some of the key aspects involved in these models, including the
impact of streptozotocin-dosage, uninephrectomy, hypertension and genetically modified strains, which can each
affect the development of disease and the interpretation of findings.

KEY WORDS: diabetic nephropathy, mouse, rat, streptozotocin.

Diabetic nephropathy is clinically defined as the progressive
development of renal insufficiency in the setting of hyperg-
lycaemia. This disease is now the major single cause of end
stage renal failure in many countries. Reliable animal
models of diabetic renal injury are a valuable tool for iden-
tifying the molecular mechanisms responsible for this
disease and for the preclinical development of new thera-
peutic strategies. Recently, a number of genetically modified
(knockout and transgenic) mouse strains have been used to
provide important insights into the roles of oxidative stress,
advanced glycation end products, inflammation and profi-
brotic mechanisms in the development of diabetic nephr-
opathy.
Chemical agents, such as streptozotocin (STZ) and
alloxan, that can selectively damage the insulin-producing
b-cells in the pancreas resulting in hyperglycaemia, are
important tools for developing animal models of diabetic
complications. These reagents can be used to study diabetic
tissue injury in most rodent strains, although the severity of
injury is partly dependent on genetic background. Models
that use STZ to induce type 1 diabetes, have been shown to
develop modest elevations in albuminuria and serum crea-
tinine and some of the histological lesions associated with
diabetic nephropathy. Obtaining meaningful data from such
Correspondence: Dr Greg Tesch, Department of Nephrology,
Monash Medical Centre, 246 Clayton Road, Clayton, Vic. 3168,
Australia. Email: greg.tesch@med.monash.edu.au
Accepted for publication 26 February 2007.
© 2007 The Authors
Journal compilation © 2007 Asian Pacific Society of Nephrology
models is dependent on various factors, including: (i) a
reliable method for establishing a consistent level of diabe-
tes; (ii) being able to maintain a steady level of diabetes
for the duration of the experiment; (iii) understanding the
disease characteristics and progression of injury in the
rodent strain being used; and (iv) the achievement of a
pathological state which has clinical relevance. In order to
assist researchers, this paper provides a description of
current protocols and key issues for developing a rodent
model of STZ-induced diabetic renal injury.
MATERIALS AND REAGENTS
The following items are required to establish a rodent model
of STZ-induced diabetes (Table 1).
METHODS
Preparation and storage of reagents
For each experiment, aliquots of STZ from the same batch are pre-
weighed into plastic microfuge tubes which are then wrapped in alu-
minium foil (to protect against light sensitivity) and stored at -20°C
with desiccant until use. Sodium citrate buffer (10 mmol/L, pH 4.5) is
prepared by dissolving 147 mg of tri-sodium citrate in 49.5 mL of
normal saline and adjusting the pH to 4.5 with approximately 0.5 mL of
1 mol/L citric acid. The citrate buffer should be used fresh or frozen in
1 mL aliquots and stored at -20°C. After thawing, each vial of frozen
citrate buffer should be used immediately and unused contents
discarded.
262
Table 1 Items required for establishing STZ-induced diabetes in rodents
Chemical reagents Equipment
Streptozotocin† Electronic weighing balance for streptozotocin (10.1 mg)
Tri-sodium citrate† Electronic weighing balance for mice (10.1 g) or rats (11 g)
Citric acid† Electronic pH meter (10.1 units)
Normal saline (0.9%) Dispensing pipette (200–1000 mL)
Isophane insulin‡ Blood glucometer and test strips§
†Available from Sigma-Aldrich, St Louis, MO, USA (website: http://www.sigma-aldrich.com). ‡Protophane, Novo Nordisk A/S, Bagsvaerd,
Denmark. §Available from Abbott Laboratories, Bedford, MA, USA.
Effect of streptozotocin on pancreatic b-cells
Streptozotocin is an analogue of N-acetylglucosamine (GlcNAc)
which is readily transported into pancreatic b-cells by GLUT-2 and
causes b-cell toxicity, resulting in insulin deficiency. STZ selectively
inhibits the activity of b-cell O-GlcNAcase, which is responsible for
removing O-GlcNAc from protein. This causes irreversible
O-glycosylation of intracellular proteins and results in b-cell apoptosis.1
Low-dose mouse model of STZ-induced diabetic
nephropathy
Based on experimental studies performed over the past decade, the
authors have formulated a reliable protocol for establishing diabetes in
mice with multiple low-dose injections of STZ.2 Male mice aged
6–7 weeks (20–25 g body weight) are fasted for 6 h prior to injection. To
induce diabetes, a microfuge tube containing preweighed STZ (ª10 mg)
is mixed immediately before use with a predetermined volume of sodium
citrate buffer to produce a final concentration of 7 mg/mL, and is
dissolved with continuous pipetting for about 5 s. This solution is then
injected intraperitoneally into each prestarved mouse at 55 mg/kg
(7.86 mL/g) using a 29G insulin needle (total volume injected = 160–
200 mL). STZ degrades quickly in aqueous solutions and should be
administered rapidly to obtain the best experimental results. Each tube
with 10 mg of STZ will provide enough solution to inject six mice. Any
remaining contents should be discarded according to the safety protocols
of the researcher’s institute. To complete the induction of disease, this
procedure must be repeated so that each mouse receives one STZ
injection for five consecutive days. This protocol normally induces a
suboptimal injury of pancreatic b-cells and progression of diabetes relies,
in part, on a secondary autoimmune insulitis.
After the completion of STZ injections, mice should be examined
for the appearance of hypoglycaemia (blood glucose <4 mmol/L) and
given glucose, if necessary. One week after the final STZ injection, mice
with a non-fasting blood glucose of less than 15 mmol/L (280 mg/dL)
should be culled, as these mice will usually not develop sufficient
diabetes to cause significant renal injury. The percentages of mice
falling into this sufficiently diabetic category will depend on the activ-
ity of the STZ and the susceptibility of the mouse strain to STZ-induced
pancreatic injury,3 and should be determined by the researcher. Gurley
et al.3 has identified an order of mouse strain susceptibility to diabetes
induced by multiple low doses of STZ (DBA/2 > C57BL/6 > MRL/
MP > 129/SvEv > BALB/c); however, it is uncertain whether this order
would apply generally to all mouse models using STZ. Using our above
protocol, the authors usually find that >90% of STZ-treated C57BL/6
mice obtain sufficient diabetes to be used in animal model studies of
diabetic nephropathy.
The US-based Animal Models of Diabetes Complications Consor-
tium (AMDCC, http://www.amdcc.org) is also proposing the adoption
of a standard low-dose model for STZ-induced diabetic complications,
GH Tesch and TJ Allen
Consumables
Plastic microfuge tubes
Aluminium foil
1 mL plastic pipette tips
Disposable plastic syringe (1 mL)
Needles for injection (27G or 29G)
which include nephropathy. In their proposed model, which is still
being finalized, mice (7–8 weeks of age) are starved for 4 h then briefly
anaesthetized with isoflurane and injected intraperitoneally with
50 mg/kg of STZ for five consecutive days. Preliminary studies using
this protocol indicate that approximately 50% of C57BL/6 mice will
develop overt diabetes after 3 weeks (see http://www.amdcc.org) with
non-fasting blood glucose levels 322 mmol/L (400 mg/dL). However, it
is likely that some investigators will consider a 50% incidence rate of
diabetes to be undesirable in terms of wastage of animals and resources.
Therefore, it is uncertain whether this protocol will be widely used.
Moderate and high-dose mouse models of STZ-induced
diabetic nephropathy
Some studies examining diabetic nephropathy in mouse strains which
are resistant to STZ-induced pancreatic injury have used either a single
high dosage of STZ (3200 mg/kg) or a two-dose regimen of STZ
(2 ¥ 100–125 mg/kg) given on consecutive days. Increasing the STZ
dosage causes greater cytotoxicity and more rapid destruction of pan-
creatic b-cells, resulting in a higher incidence and severity of diabetes.
However, at high doses, STZ has a non-specific cytotoxicity effect
which has been shown to cause acute kidney damage in mice and rats.4,5
Consequently, models using high doses of STZ can develop a nephr-
opathy which results from hyperglycaemia-induced injury superim-
posed on acute renal STZ-cytotoxicity, making it difficult to interpret
any findings.4
The following protocol describes a two-dose procedure
(2 ¥ 125 mg/kg per day STZ) for establishing diabetes in C57BL/6 mice
with genetic deficiencies which facilitate mild resistant to STZ.6 Renal
injury in this model does not appear to be associated with acute tubular
cytotoxicity, based on the ability of insulin treatment to prevent renal
pathology.6 An aliquot of STZ (10–15 mg/tube) is dissolved immedi-
ately before use with a predetermined volume of sodium citrate buffer to
produce a final concentration of 15.6 mg/mL. This solution is then
injected intraperitoneally into each mouse at 125 mg/kg (8 mL/g). The
same procedure is repeated for each mouse at 24 h after the first injec-
tion. Using this procedure, approximately 90% of wild type C57BL/6
mice will develop overt diabetes within 2 weeks, with a lower incidence
expected for more resistant genotypes.6 Because the pancreatic injury is
more severe in this model, the diabetic mice will need to be monitored
for severe hyperglycaemia (blood glucose >33 mmol/L, 600 mg/dL) and
administered isophane insulin (see section Animal Welfare and Mainte-
nance) to reduce blood glucose to a more tolerable range (16–
33 mmol/L, 300–600 mg/dL).
Rat models of STZ-induced diabetic nephropathy
Models of STZ-induced diabetic nephropathy are commonly performed
in Sprague-Dawley (SD), Wistar-Kyoto (WKY) or spontaneously
© 2007 The Authors
Journal compilation © 2007 Asian Pacific Society of Nephrology
Streptozotcin-induced diabetic nephropathy in rodents
hypertensive (SHR) rats. In these models, male rats at 8 weeks of age
(200–250 g) are starved for 16 h and injected once into the tail vein
with STZ (SD = 55 mg/kg, WKY = 60 mg/kg, SHR = 45 mg/kg) in
sodium citrate buffer (1 mL/kg).7,8 STZ has also been administered
intraperitoneally to rats, however, this is less common as intravenous
injections are relatively easy to perform in rats and give more consistent
results. In addition, the STZ dosage required to achieve diabetes via an
intraperitoneal route is expectedly higher.
Following the STZ injection, rats should be given drinking water
supplemented with sucrose (15 g/L) for 48 h, to limit early mortality as
stores of insulin are released from damaged pancreatic islets. At 1 week
after STZ, rats should be assessed for hyperglycaemia and those with
fasting blood glucose of over 15 mmol/L (280 mg/dL), which is usually
around 90%, should be included in studies of diabetic nephropathy. To
prevent subsequent development of ketonuria, diabetic rats should be
given daily subcutaneous injections of long-acting insulin (2–4 U/rat,
Protophane, Novo Nordisk Industries A/S, Bagsvaerd, Denmark) to
maintain blood glucose levels in a desirable range (16–33 mmol/L,
300–600 mg/dL).9 Studies examining the effects of treatments on the
development of diabetic nephropathy should not be started until at
least 3 weeks after STZ when the kidneys have recovered from the
acute mild nephrotoxic effects of STZ.5
Following induction of diabetes with STZ, the development of
renal injury is accelerated and becomes more profound in SHR com-
pared with normotensive rats (WKY).8 Vascular hypertension activates
the renin-angiotensin system which alters renal haemodynamics,
increases glomerular basement membrane thickness and promotes the
development of inflammation and fibrosis in the setting of renal
injury.10 A long-term study of STZ-induced diabetic nephropathy
involving SHR has shown that the urine albumin excretion rate
(UAER) is threefold higher in diabetic SHR (149 1 1 mg/24 h) at
32 weeks compared with control SHR (49 1 1 mg/24 h).9
Uninephrectomized rat model of streptozotocin-induced
diabetic nephropathy
Models of STZ-induced diabetic nephropathy have also been per-
formed in different rat strains (SD, Wistar, SHR) following uninephre-
ctomy, which is thought to accelerate the progression of renal injury.
Uninephrectomy results in enlargement of the remaining kidney,
which is further increased by the development of diabetes. Uninephre-
ctomy has been shown to increase glomerular capillary pressure in SHR
rats which promotes diabetic glomerular injury.11 However, interpreta-
tion of this model is complex, as it is difficult to dissect the relative
contributions of STZ-induced hyperglycaemia and uninephrectomy-
induced changes in glomerular haemodynamics in the development of
renal injury. In a study by Utimura et al.12 male Wistar rats (ª250 g)
were uninephrectomized (right nephrectomy) during anaesthesia
(50 mg/kg intraperitoneal sodium pentobarbital) and allowed to
recover from surgery (3 weeks). They were then made diabetic by a
single intravenous injection of STZ (65 mg/kg) and blood glucose
assessed 2 days later. The blood glucose was then maintained between
16 and 22 mmol/L (300–400 mg/dL) for the next 8 months with insulin
treatment. These uninephrectomized diabetic rats achieved a UAER of
approximately 60 mg/24 h at 8 months which was nearly three times
higher than non-diabetic control rats at the same age.
Biochemical assessment of diabetes
Evaluation of rodent hyperglycemia is routinely performed by obtaining
a drop of blood from the tail vein, placing it on a test strip, and
measuring the glucose level with a standard patient glucometer.
However, more accurate readings can be obtained by automated
© 2007 The Authors
Journal compilation © 2007 Asian Pacific Society of Nephrology
263
glucose-oxidase assays performed in biochemistry labs. To reduce the
variations in blood glucose readings associated with feeding habits,
blood glucose should be measured on animals after a standard fasting
period at a designated time of day. This fasting time typically varies
between 3 and 6 h among research groups performing mouse studies.
However, fasting is not routinely performed before blood glucose mea-
surements in rat models of STZ-induced diabetic nephropathy. For a
more comprehensive measurement of average blood glucose levels,
heparinized tail vein blood (ª25 mL) can be collected from rodents and
assessed to determine the percentage of glycated haemaglobin. This
assay is routinely performed by HPLC in hospital pathology labs.
Because the blood cell turnover for rodents is approximately 30 days, a
glycated haemaglobin reading provides an indirect assessment of the
average blood glucose level over the previous month. Glycated haema-
globin levels greater than 7% have led to significant renal lesions in
diabetic mouse kidneys.
Biochemical assessment of renal injury
Urine albumin excretion is considered to be one of the most sensitive
markers of renal injury. Normal mice have a UAER of approximately
10 mg/day. Studies of STZ-treated mice with a C57BL/6 background
have detected a modest increase in UAER of 30–90 mg/day after
18–20 weeks with the highest levels being observed in hyperlipidaemic
ApoE deficient mice.13 Measurements of UAER normally requires
rodents to be maintained in metabolic cages for 24 h to collect urine.
The urine volume is measured and aliquots stored frozen for subsequent
measurement of albumin by enzyme-linked immunosorbent assay
(ELISA). Previous studies have successfully used radioimmunoassay for
assessing albuminuria;10 however, this technique is time-consuming.
Reliable ELISA kits are now available from Bethyl Laboratories (Mont-
gomery, TX, USA, http://www.bethyllabs.com) for measuring mouse and
rat albumin and from Exocell (Philadelphia, PA, USA, http://
www.exocell.com) for mouse albumin. The albumin : creatinine ratio in
urine can also be used to measure diabetic renal injury in rodents. This
technique can be applied when metabolic cages are not available or if
there is concern about the potential stress imposed on mice housed in
metabolic cages. For these measurements, urine is collected by briefly
allowing animals to urinate into a Petri dish. Urine creatinine can be
assessed by a common colourimetric assay (picric-acid-Jaffe method), an
enzymatic assay or an HPLC method.14
Renal function is most commonly assessed by calculating creatinine
clearance as a measure of glomerular filtration rate (GFR). This
involves obtaining creatinine measurements in serum or plasma and in
a 24 h urine collection. This analysis has been traditionally performed
in rodent models of renal disease using the picric-acid-Jaffe method.
However, recent studies indicate that rodent serum or plasma creati-
nine values are overestimated using the Jaffe method, because of inter-
ference from haemaglobin and possibly other factors. In contrast, an
enzymatic method (CREA plus, Roche Diagnostics, Mannheim,
Germany) using creatininase has been shown to produce measurements
of mouse plasma creatinine which correlate with HPLC values when
samples show no visible haemolysis.14 Therefore, analysis of creatinine
clearance in rodent models of diabetic kidney disease should be per-
formed using reliable techniques such as HPLC or a creatininase assay.
An alternative approach for determining GFR is to measure clear-
ance of labelled inulin or diethylene triamine penta-acetic acid
(DTPA). Inulin clearance measurements have been achieved in rats
and mice by surgical intraperitoneal implantation of osmotic
minipumps (Alza Corporation, Palo Alto, CA, USA) which are filled
with FITC-conjugated inulin (Sigma, St Louis, MO, USA) that is
released at a steady state.15 After implantation, urine from a 24 h
collection and plasma are assessed for levels of FITC-inulin by fluorom-
etry. The GFR based on clearance of inulin or creatinine is calculated
264
by the amount excreted in urine divided by the plasma concentration
and is usually expressed as mL/min per g body weight in rodents. GFR
measurements have also been determined in rodents by a single tail
vein injection of 99mtechnetium-labelled DTPA (99mTc-DTPA).16 In
this technique, GFR is calculated by measuring plasma radioactivity at
a specified time after injection (43 min) which is then compared with
a reference made at the time of injection.
Assessment of renal pathology
Renal pathology in diabetic rodent kidneys can be routinely assessed on
tissue sections stained with periodic acid Schiff ’s reagent and Harris
haematoxylin. Kidney cross-sections 3–4 mm thick are fixed in 10%
neutral buffered formalin for 2–3 h and then processed for paraffin
embedding. In order to best preserve kidney morphology, some groups
also perfuse the kidneys with fixative prior to removal and dissection,
however, this procedure is not routinely used in literature. After pro-
cessing, tissue sections 2–3 mm thick are attached to slides, dewaxed
and stained with periodic acid Schiff’s reagent followed by haematoxy-
lin according to standard textbook protocols. Microscope images of
these sections can be used in the analysis of glomerular hypertrophy,
glomerular and interstitial hypercellularity, tubular dilatation and
atrophy, and interstitial volume.17
Additional pathological characterization can be performed by a
number of different techniques. Electron microscopy is classically used to
assess morphological changes including thickening of the glomerular
basement membrane and mesangial expansion.10 Total collagen deposi-
tion, using text book histochemical stains such as Sirius Red or Masson
Trichrome, can be used to evaluate fibrosis. Also, specific collagens,
myofibroblast accumulation or inflammatory cells can be identified by
immunostaining which is usually best performed on sections fixed in
paraformaldehyde.17
Assessment of hypertension
Although the progression of human diabetic nephropathy is strongly
associated with hypertension, the blood pressure changes seen in STZ-
induced diabetic rodent models is usually mild unless the strain being
used is spontaneously hypertensive. Hypertension is routinely measured
by indirect tail-cuff plethysmography in non-anaesthetized rodents, and
requires the averaging of repeated measurements at selected time-
points.18 This technique is particularly difficult in mice which need a lot
of training to become familiar to the procedure without causing addi-
tional stress. More recently, radio telemetry has allowed continuous
direct blood pressure monitoring in studies involving conscious rodents
by inserting a radio-implant into an artery.9 Both of these methods have
been used to evaluate the effects of antihypertensive treatments on the
progression of STZ-induced diabetic nephropathy. The equipment used
is relatively expensive and the procedures involved require a significant
amount of training to be sufficiently skilled, however, the high sensi-
tivity of these techniques can lead to results which provide important
insight into therapeutic applications and disease mechanisms.
ADDITIONAL KEY ISSUES FOR
EXPERIMENTAL DESIGN
Animal maintenance and welfare
When designing experiments in animal models of diabetic
nephropathy, it is important to predetermine protocols for
animal monitoring and criteria for intervention. This will
help avoid animal wastage. Severely diabetic rodents can
GH Tesch and TJ Allen
suffer from weight loss, dehydration, cataracts, lethargy and
diabetic coma. Diabetic animals should be visually moni-
tored at regular intervals (2–3 times weekly) and assessed for
health status using a checklist with specific scoring criteria
(see example by David B. Morton at http://dels.nas.edu/
ilar_n/ilarjournal/41_2/Systematic.shtml). Rodents with
suspected welfare problems should be examined more often,
including measurements of food and water intake. Guidance
for rodent monitoring, appropriate treatment or humane
euthanasia can usually be obtained from journals (http://
www.lal.org.uk), animal welfare committees, veterinarians
and trained animal technicians. Rodents with non-fasting
blood glucose levels between 16 and 30 mmol/L can nor-
mally be maintained without intervention. Rodents with a
non-fasting blood glucose above 35 mmol/L require insulin
treatment to avoid weight loss and those below 4 mmol/L
require administration of glucose or glucagon to avoid dia-
betic coma. Insulin treatment to lower blood glucose into a
manageable range is best achieved by subcutaneous injec-
tion of a suboptimal dose of long-acting isophane insulin
(e.g. Protophane). The insulin dose required will vary with
species, strain and disease severity and should be determined
by the researcher. Subcutaneous implants which continu-
ously release insulin are less reliable and often result in
episodes of hypoglycaemia and diabetic coma. Liquid nutri-
tion supplements (e.g. Ensure, Abbott Laboratories) can
help in preventing weight loss in severely diabetic animals
when combined with insulin treatment.
Creating and validating a new model of STZ-induced
diabetic nephropathy
In order to create a reliable model of STZ-induced diabetic
nephropathy, a number of preliminary findings need to be
established with each rodent strain being used. Gender and
genetic background will affect the susceptibility of rodents
to STZ-induced pancreatic injury and to the development
of diabetic renal injury. Male rodents are generally more
susceptible to the effects of STZ and tend to develop greater
hyperglycaemia. In addition, some strains of rodents are
more hypertensive than others and will develop a more
profound renal injury after the onset of diabetes. Recently,
Qi et al.19 evaluated the development of STZ-induced
diabetic nephropathy in various mouse strains with hyperg-
lycaemia. This study showed that the level of hyperglycae-
mia alone was unable to account for the differences between
strains in the severity of renal injury. When compared with
the commonly used C57BL/6 strain, DBA/2J and KK/H1J
mice were found to develop increased albuminuria and
greater severity in renal morphological changes, including
mesangial expansion, nodular glomerulosclerosis and arteri-
olar hyalinosis. Therefore, choosing the appropriate strain
and gender of rodents should be considered carefully.
When determining the effects of specific molecules in
genetically modified strains (knockouts or transgenics), it
is particularly important to make sure that the genetically
modified rodents are only different to the wild type con-
trols in the gene of interest. The appropriate dose of STZ
© 2007 The Authors
Journal compilation © 2007 Asian Pacific Society of Nephrology
Streptozotcin-induced diabetic nephropathy in rodents
required to induce a sustainable diabetes in >50% of
rodents, without inducing direct renal injury, should be
determined in both wild type and genetically modified
strains. The incidence of diabetes obtained with the same
STZ dose may vary between these strains. For example,
the authors have previously found that a STZ dose induc-
ing a >90% incidence of diabetes in wild type C57BL/6
mice produced only a 60% incidence of diabetes in MCP-1
deficient C57BL/6 mice.6 Therefore, the results of dose-
seeking studies in each strain should be considered
together in selecting the single most appropriate dosage of
STZ to be used in a major study which compares strains. A
pilot study, with the selected dose of STZ, should then be
performed in order to establish a time course of diabetic
renal injury and choose appropriate endpoints. This infor-
mation can then be used to design a major study and also
determine appropriate points for potential therapeutic
intervention.
In humans, diabetic nephropathy is characterized clini-
cally by the development of microalbuminuria, which
progresses to macro-albuminuria and a decline in renal
function. These clinical features are also seen in rodent
models of STZ-induced diabetic nephropathy, although
the level of albuminuria and the loss of renal function
are much less severe. The major histological findings in
human diabetic nephropathy are glomerular basement
membrane thickening by electron microscopy in the
absence of immune deposits, mesangial expansion and
sclerosis with or without the development of nodular
mesangial sclerosis (i.e. Kimmelstiel–Wilson nodules),
tubulointerstitial fibrosis and arteriolar hyalinosis. These
features, with the exception of Kimmelstiel–Wilson
nodules, have also been detected in rodent models of STZ-
induced diabetic nephropathy, although their severity in
rodents is usually milder.
Based on present knowledge of human diabetic nephr-
opathy, the AMDCC is currently proposing that a desirable
rodent model of diabetic renal disease should include the
following components: (i) a greater than 50% decline in
GFR over the lifetime of the animal; (ii) a 3100-fold
increase in the UAER compared with controls of the same
strain, age and gender; and (iii) histopathology findings
which include mesangial sclerosis (a 50% increase in mesan-
gial volume), any degree of arteriolar hyalinosis, glomerular
basement membrane thickening (a >25% increase com-
pared with baseline by electron microscopy morphometry)
and tubulo-interstitial fibrosis.
Currently, there are no mouse or rat models which
achieve the first two criteria as a result of diabetes, however,
a number of studies have shown significant histopatho-
logical lesions which achieve or approach the histological
criteria. Such models have already proved useful in our
understanding of the mechanisms of diabetic renal disease
and, often, the conclusions are supported by clinical and
biopsy findings in human patients. Future developments in
STZ-induced models of diabetic nephropathy, perhaps
involving novel rodent strains, may provide the additional
conditions necessary to achieve all the recommended crite-
ria defined by the AMDCC.
© 2007 The Authors
Journal compilation © 2007 Asian Pacific Society of Nephrology
265
DISCUSSION
Although the use of STZ is a robust method for inducing
diabetes in rodents, the development of diabetic nephropa-
thy in these animals shows limited resemblance to the
human disease, presumably because of physiological, meta-
bolic and hormonal differences. Consequently, extensive
genetic manipulation may be required to engineer more
appropriate rodent models of diabetic nephropathy.
Genetic modified rodents have recently been used to
create models of STZ-induced diabetic nephropathy with
increased renal injury. These models are useful for testing
novel therapies which target disease mechanisms. Mice
which are genetically deficient in apolipoprotein-E (ApoE)
have a reduced ability to clear plasma lipoproteins,13 which
results in increased circulating levels of cholesterol and
triglycerides. These ApoE–/– mice are more susceptible to
vascular injury and, consequently, diabetic complications
progress more rapidly in an ApoE deficient strain com-
pared with equally diabetic wild type mice of the same
background strain. Models of STZ-induced diabetic
nephropathy in ApoE–/– mice have been used to examine
the effects of PPAR-a and PPAR-g agonists and specific
tyrosine kinase inhibitors as potential intervention treat-
ments.20,21 Hypertensive transgenic (mREN-2)27 rats
which have tissue overexpression of renin develop a
more severe renal injury than either normotensive or
SHR strains following induction of diabetes with STZ.21
Diabetic (mRen-2)27 rats have a greater than 50%
decline in GFR with nodular and diffuse glomerulosclerosis
reminiscent of diabetic nephropathy.22 This rat model has
been used to examine the therapeutic benefits of antihy-
pertensive agents and inhibitors of advanced glycation
end products, specific kinases and transforming growth
factor-b. However, a recent article suggests that long-term
studies in this model may more closely resemble severe
hypertensive nephrosclerosis than progressive diabetic
nephropathy.23
In conclusion, the extensive use of STZ to create
models of diabetic nephropathy indicates that this tech-
nique is an important and widely accepted tool for
examining the mechanisms of diabetic renal injury and
potential therapeutic interventions. In order to better
compare and interpret findings obtained from different
experiments performed around the world, it will be ben-
eficial to obtain some general agreement on protocols for
establishing and analysing models of STZ-induced diabetic
nephropathy in specified rodent strains. It is hoped that
information presented in this manuscript will help in
developing such an agreement.
ACKNOWLEDGEMENTS
GHT is supported by a Career Development Award from the
National Health and Medical Research Council of Austra-
lia, Kidney Health Australia and the Australian and New
Zealand Society of Nephrology. TJA is a recipient of a
Career Development Award/RD Wright Fellowship from
the National Health and Medical Research Council of
266
Australia and Diabetes Australia. Animal model studies
were supported by a Einstein Juvenile Diabetes Research
Foundation Centre grant.
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© 2007 The Authors
Journal compilation © 2007 Asian Pacific Society of Nephrology