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Methods in Renal Research

Rodent models of streptozotocin-induced diabetic nephropathy

GREG H TESCH1,2 and TERRI J ALLEN3

Departments of  1 Nephrology 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 1diabetes 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 susceptibilityin the development of diabetic nephropathy. In this report, the authors describe and compare the current protocolsbeing used to establish models of diabetic nephropathy in rat and mouse strains using streptozotocin. Theauthors also list some of the histological criteria and biochemical measurements which are being used to validatethese models. In addition, our review explains some of the key aspects involved in these models, including theimpact 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 progressivedevelopment of renal insufficiency in the setting of hyperg-lycaemia. This disease is now the major single cause of endstage renal failure in many countries. Reliable animalmodels 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 toprovide 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) andalloxan, that can selectively damage the insulin-producingb-cells in the pancreas resulting in hyperglycaemia, areimportant tools for developing animal models of diabeticcomplications. These reagents can be used to study diabetictissue injury in most rodent strains, although the severity of injury is partly dependent on genetic background. Modelsthat use STZ to induce type 1 diabetes, have been shown todevelop modest elevations in albuminuria and serum crea-tinine and some of the histological lesions associated withdiabetic nephropathy. Obtaining meaningful data from such

models is dependent on various factors, including: (i) areliable method for establishing a consistent level of diabe-tes; (ii) being able to maintain a steady level of diabetesfor the duration of the experiment; (iii) understanding thedisease characteristics and progression of injury in therodent strain being used; and (iv) the achievement of a

pathological state which has clinical relevance. In order toassist researchers, this paper provides a description of current protocols and key issues for developing a rodentmodel of STZ-induced diabetic renal injury.

MATERIALS AND REAGENTS

The following items are required to establish a rodent modelof 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.

Correspondence: Dr Greg Tesch, Department of Nephrology,

Monash Medical Centre, 246 Clayton Road, Clayton, Vic. 3168,

Australia. Email: greg.tesch@med.monash.edu.auAccepted for publication 26 February 2007.

© 2007 The Authors  Journal compilation © 2007 Asian Pacific Society of Nephrology

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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 diabeticnephropathy

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 bodyweight) arefastedfor 6 h prior to injection. To

induce diabetes, a microfugetube containing preweighed STZ (ª10 mg)

is mixed immediately before usewith a predeterminedvolumeof 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–

200m

L). 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 shouldbe discarded according to the safetyprotocols

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 injuryof pancreaticb-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 thesusceptibility 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,

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-induceddiabetic 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 incidenceexpected 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

Table 1 Items required for establishing STZ-induced diabetes in rodents

Chemical reagents Equipment Consumables

Streptozotocin† Electronic weighing balance for streptozotocin (10.1 mg) Plastic microfuge tubesTri-sodium citrate† Electronic weighing balance for mice (10.1 g) or rats (11 g) Aluminium foilCitric acid† Electronic pH meter (10.1 units) 1 mL plastic pipette tips

  Normal saline (0.9%) Dispensing pipette (200–1000mL) Disposable plastic syringe (1 mL)

Isophane insulin‡ Blood glucometer and test strips§ Needles for injection (27G or 29G)

†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.

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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 (491 1 mg/24 h).9

Uninephrectomized rat model of streptozotocin-induceddiabetic 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 between16 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

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 measuringmouse and

rat albumin and from Exocell (Philadelphia, PA, USA, http://

www.exocell.com)for mouse albumin. Thealbumin : 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 commoncolourimetric 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 whensamples 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

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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

numberof different techniques.Electronmicroscopyis classicallyused 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 FOREXPERIMENTAL DESIGN

Animal maintenance and welfare

When designing experiments in animal models of diabeticnephropathy, it is important to predetermine protocols foranimal monitoring and criteria for intervention. This willhelp avoid animal wastage. Severely diabetic rodents can

suffer from weight loss, dehydration, cataracts, lethargy anddiabetic coma. Diabetic animals should be visually moni-tored at regular intervals (2–3 times weekly) and assessed forhealth 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 withsuspected welfare problems should be examined more often,

including measurements of food and water intake. Guidancefor rodent monitoring, appropriate treatment or humaneeuthanasia can usually be obtained from journals (http://www.lal.org.uk), animal welfare committees, veterinariansand trained animal technicians. Rodents with non-fastingblood glucose levels between 16 and 30 mmol/L can nor-mally be maintained without intervention. Rodents with anon-fasting blood glucose above 35 mmol/L require insulintreatment to avoid weight loss and those below 4 mmol/Lrequire administration of glucose or glucagon to avoid dia-betic coma. Insulin treatment to lower blood glucose into amanageable 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 withspecies, strain and disease severity and should be determinedby the researcher. Subcutaneous implants which continu-ously release insulin are less reliable and often result inepisodes of hypoglycaemia and diabetic coma. Liquid nutri-tion supplements (e.g. Ensure, Abbott Laboratories) canhelp in preventing weight loss in severely diabetic animalswhen combined with insulin treatment.

Creating and validating a new model of STZ-induceddiabetic nephropathy

In order to create a reliable model of STZ-induced diabetic

nephropathy, a number of preliminary findings need to beestablished with each rodent strain being used. Gender andgenetic background will affect the susceptibility of rodentsto STZ-induced pancreatic injury and to the developmentof diabetic renal injury. Male rodents are generally moresusceptible to the effects of STZ and tend to develop greaterhyperglycaemia. In addition, some strains of rodents aremore hypertensive than others and will develop a moreprofound renal injury after the onset of diabetes. Recently,Qi et al.19 evaluated the development of STZ-induceddiabetic 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 withthe commonly used C57BL/6 strain, DBA/2J and KK/H1Jmice were found to develop increased albuminuria andgreater severity in renal morphological changes, includingmesangial expansion, nodular glomerulosclerosis and arteri-olar hyalinosis. Therefore, choosing the appropriate strainand gender of rodents should be considered carefully.

When determining the effects of specific molecules ingenetically modified strains (knockouts or transgenics), itis particularly important to make sure that the geneticallymodified rodents are only different to the wild type con-trols in the gene of interest. The appropriate dose of STZ

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required to induce a sustainable diabetes in >50% of rodents, without inducing direct renal injury, should bedetermined in both wild type and genetically modifiedstrains. The incidence of diabetes obtained with the sameSTZ 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-1deficient C57BL/6 mice.6 Therefore, the results of dose-seeking studies in each strain should be consideredtogether in selecting the single most appropriate dosage of STZ to be used in a major study which compares strains. Apilot study, with the selected dose of STZ, should then beperformed in order to establish a time course of diabeticrenal injury and choose appropriate endpoints. This infor-mation can then be used to design a major study and alsodetermine appropriate points for potential therapeuticintervention.

In humans, diabetic nephropathy is characterized clini-cally by the development of microalbuminuria, which

progresses to macro-albuminuria and a decline in renalfunction. These clinical features are also seen in rodentmodels of STZ-induced diabetic nephropathy, althoughthe level of albuminuria and the loss of renal functionare much less severe. The major histological findings inhuman diabetic nephropathy are glomerular basementmembrane thickening by electron microscopy in theabsence of immune deposits, mesangial expansion andsclerosis with or without the development of nodularmesangial sclerosis (i.e. Kimmelstiel–Wilson nodules),tubulointerstitial fibrosis and arteriolar hyalinosis. Thesefeatures, with the exception of Kimmelstiel–Wilsonnodules, 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 desirablerodent model of diabetic renal disease should include thefollowing components: (i) a greater than 50% decline inGFR over the lifetime of the animal; (ii) a 3100-foldincrease in the UAER compared with controls of the samestrain, age and gender; and (iii) histopathology findingswhich include mesangial sclerosis (a 50% increase in mesan-gial volume), any degree of arteriolar hyalinosis, glomerularbasement 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 whichachieve 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 histologicalcriteria. Such models have already proved useful in ourunderstanding of the mechanisms of diabetic renal diseaseand, often, the conclusions are supported by clinical andbiopsy findings in human patients. Future developments inSTZ-induced models of diabetic nephropathy, perhapsinvolving novel rodent strains, may provide the additionalconditions necessary to achieve all the recommended crite-ria defined by the AMDCC.

DISCUSSION

Although the use of STZ is a robust method for inducingdiabetes in rodents, the development of diabetic nephropa-thy in these animals shows limited resemblance to thehuman disease, presumably because of physiological, meta-bolic and hormonal differences. Consequently, extensive

genetic manipulation may be required to engineer moreappropriate rodent models of diabetic nephropathy.

Genetic modified rodents have recently been used tocreate models of STZ-induced diabetic nephropathy withincreased renal injury. These models are useful for testingnovel therapies which target disease mechanisms. Micewhich are genetically deficient in apolipoprotein-E (ApoE)have a reduced ability to clear plasma lipoproteins,13 whichresults in increased circulating levels of cholesterol andtriglycerides. These ApoE–/– mice are more susceptible tovascular injury and, consequently, diabetic complicationsprogress more rapidly in an ApoE deficient strain com-pared with equally diabetic wild type mice of the same

background strain. Models of STZ-induced diabeticnephropathy in ApoE–/– mice have been used to examinethe effects of PPAR-a and PPAR-g agonists and specifictyrosine kinase inhibitors as potential intervention treat-ments.20,21 Hypertensive transgenic (mREN-2)27 ratswhich have tissue overexpression of renin develop amore severe renal injury than either normotensive orSHR 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 glomerulosclerosisreminiscent of diabetic nephropathy.22 This rat model hasbeen used to examine the therapeutic benefits of antihy-pertensive agents and inhibitors of advanced glycationend products, specific kinases and transforming growth

factor-b. However, a recent article suggests that long-termstudies in this model may more closely resemble severehypertensive nephrosclerosis than progressive diabeticnephropathy.23

In conclusion, the extensive use of STZ to createmodels of diabetic nephropathy indicates that this tech-nique is an important and widely accepted tool forexamining the mechanisms of diabetic renal injury andpotential therapeutic interventions. In order to bettercompare and interpret findings obtained from differentexperiments performed around the world, it will be ben-eficial to obtain some general agreement on protocols forestablishing and analysing models of STZ-induced diabeticnephropathy in specified rodent strains. It is hoped thatinformation presented in this manuscript will help indeveloping 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 NewZealand Society of Nephrology. TJA is a recipient of aCareer Development Award/RD Wright Fellowship fromthe National Health and Medical Research Council of 

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Australia and Diabetes Australia. Animal model studieswere supported by a Einstein Juvenile Diabetes ResearchFoundation Centre grant.

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GH Tesch and TJ Allen266

© 2007 The Authors

  Journal compilation © 2007 Asian Pacific Society of Nephrology