1Medical Research Laboratories, Institute of Experimental Clinical Research, Aarhus University Hospital, 2Department of Cell Biology, Institute of Anatomy, Aarhus University, Aarhus, Denmark, 3Renal Unit, Department of Internal Medicine, Gent University Hospital, 4Department of Physiology and Physiopathology, Gent University, Gent and 5Renal Unit, Department of Internal Medicine, AZ Sint-Jan AV, Brugge, Belgium
Correspondence and offprint requests to: Bieke Schrijvers, Renal Unit, Gent University Hospital, Gent, Belgium. Email: Bieke.Schrijvers{at}UGent.be
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Abstract |
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Methods. Urinary albumin excretion, creatinine clearance and blood pressure were measured at the age of 2, 8 and 14 months in 12 female GotoKakizaki rats and 10 female, non-diabetic Wistar rats. To study kidney morphology, kidney weight, glomerular volume, basement membrane thickness, mesangial fraction and total mesangial volume were determined at 14 months.
Results. Urinary albumin excretion rose progressively over time in both groups, but was significantly higher in GotoKakizaki rats than in Wistar rats. Creatinine clearance decreased over time in GotoKakizaki rats but not in Wistar rats. Blood pressure was in the normotensive range in all animals throughout the study. Kidney weight, glomerular volume, basement membrane thickness, mesangial fraction and total mesangial volume were significantly higher in GotoKakizaki rats than in Wistar rats. Body weight and blood glucose levels were higher, whereas serum insulin levels were not different or lower in GotoKakizaki rats compared with Wistar rats.
Conclusion. The GotoKakizaki rat is a lean, hyperglycaemic, euinsulinaemic, normotensive experimental model of type 2 diabetes with robust functional and structural renal changes.
Keywords: albuminuria; basement membrane thickness; glomerular volume; GotoKakizaki rat; kidney; mesangium
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Introduction |
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The GotoKakizaki rat has been presented as a model for type 2 diabetes with hyperglycaemia in the absence of obesity or hypertension. The GotoKakizaki rat has been bred from non-diabetic Wistar rats selected from a normal population with a glucose tolerance test slightly deviating from the normal range [2]. Few studies on renal changes in GotoKakizaki rats have been published, and those included small numbers of exclusively male animals [36]. The aim of this study was to describe the long-term renal morphological and some functional changes in the female GotoKakizaki rat, in order to evaluate its suitability as a model for renal disease in human type 2 diabetes.
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Materials and methods |
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At 2, 8 and 14 months, body weight and food consumption were measured in all rats. Blood glucose was determined on tail vein blood. Under light ether anaesthesia, blood samples were drawn from the retro-orbital venous plexus using heparinized capillary tubes. After centrifugation, serum samples were stored at 80°C for later determination of serum insulin, fructosamine and 8-isoprostane (8-epi PGF2) levels. An intraperitoneal glucose tolerance test (IPGTT) was performed in six rats of each group. The animals were placed in metabolic cages to collect 24-h urine samples, which were stored at 80°C for later analysis of urinary albumin excretion (UAE).
As previously described, systolic blood pressure (BP) was recorded in the early afternoon by the tail-cuff method on awake rats after they were accustomed to rest quietly in warmed plexi-glass restrainers [7]. For each animal, the blood pressure level was determined as the mean value of 10 consecutive measurements. At 14 months, the rats were anaesthetized with pentobarbital (50 mg/kg i.p.), and non-fasting blood samples were taken from the retro-orbital venous plexus.
Right and left kidney were quickly removed, carefully cleaned and weighed. The middle piece of the right kidney (including the papilla) was fixed in a 0.1 M cacodylate buffer with 2% paraformaldehyde and 1% glutaraldehyde, for electron microscopic determination of basement membrane thickness (BMT) and mesangial fraction, and the middle piece of the left kidney (including the papilla) was fixed in 4% paraformaldehyde for light microscopic measurement of glomerular volume. Liver and heart were removed and weighed. In each group, one animal was excluded from the study due to unexplained massive body weight loss.
Metabolic measurements
Blood and urinary glucose concentration
Blood glucose was measured in tail vein blood by Haemo-Glucotest 1-44 (Roche Diagnostics Scandinavia AB, Copenhagen, Denmark) and Reflolux II reflectance meter (Boehringer-Mannheim, Mannheim, Germany), and urine was tested for ketone bodies by Neostix-4 (Ames, Stoke Poges, Slough, UK).
IPGTT
An IPGTT was performed by i.p. injection of a 20% glucose solution in a dose of 2 g/kg body weight. Animals were tested at 1.00 p.m. after a 6 h fast. Blood glucose was measured by Haemo-Glucotest 1-44 in tail vein blood prior to, and 30, 60, 90, 120 and 150 min after the glucose administration.
Serum insulin levels
Serum insulin levels were determined using an ultrasensitive Rat Insulin ELISA Kit (DRG Diagnostics, Marburg, Germany). The intra- and inter-assay coefficients of variation were <5 and <10%, respectively.
Serum fructosamine levels
The fructosamine assay (Fructosamine Test Plus, Hoffman-La Roche, Basle, Switzerland) was performed as previously described [8]. The principle of the assay relies on the reducing potential of ketoamines in alkaline medium. The serum fructosamine assay measures all glycated serum proteins by forming the corresponding eneaminols, which in turn reduce nitroblue tetrazolium to the coloured formazan derivative. The rate of formazane colour development correlates with the fructosamine level. The intra- and inter-assay coefficients of variation were <5 and <10%, respectively.
Serum 8-epi PGF2 levels
Total serum 8-epi PGF2 levels were determined using an 8-Isoprostane EIA Kit (Cayman Chemical Company, Ann Arbor, MI) according to the manufacturer's instructions. For sample purification by a solid phase extraction method, a 250 µl serum sample was diluted with 500 µl of ethanol. After centrifugation, and incubation of the supernatant with 15% KOH, the sample was diluted with Ultra Pure H2O to a total volume of 2.5 ml. In the final step of the purification protocol, 8-epi PGF2
was eluated with 1.25 ml ethylacetate with 1% methanol. All samples were run in the same assay. The intra-assay coefficient of variation was <5%.
Renal measurements
UAE and creatinine clearance (CCr)
As previously described, the urinary albumin concentration in 24-h urine collections was determined by an in-house rat albumin radioimmunoassay, using rabbit anti-rat albumin antibody RARa/Alb (Nordic Pharmaceuticals and Diagnostics, Tilburg, The Netherlands), and globulin-free rat albumin for standard and iodination (Sigma Chemical Co., St Louis, MO) [9]. Serum and urinary creatinine concentrations were measured by an automated technique adapted from the method of Jaffé and corrected for the prevailing glucose contents interfering in the Jaffé reaction. The CCr was expressed in ml/min. The intra- and inter-assay coefficients of variation were <5 and <10%, respectively, for both assays.
Estimation of glomerular volume
The middle part of the left kidney (containing the papilla) was embedded in paraffin for light microscopic examination. Sections of 2 µm thickness were cut on a rotation microtome (Leica Rotation Microtome RM 2165, Leica, Vienna, Austria) and stained with periodic acidSchiff and haematoxylin. The thickness of the sections was controlled routinely by a Digital Microcator ND 221 (Heidenhain, Traunreut, Germany) attached to the microscope. In each animal, the mean glomerular tuft volume (VG) was determined from the mean glomerular cross-sectional area (AG) in an Olympus BX51TF light microscope (Olympus Co., Tokyo, Japan) at a magnification of 420x as previously described [10,11]. The areas were determined with a 2D version of the nucleator (CAST, Olympus, Denmark) by light microscopy as the average area of a total of 80100 glomerular profiles (i.e. capillary tuft omitting the proximal tubular tissue and the Bowmann capsule) [10]. VG was calculated as: VG = ß/k x (AG)3/2, where ß = 1.38, which is the shape coefficient for spheres (the idealized shape of glomeruli), and k = 1.1, which is a size distribution coefficient [11].
Estimation of mesangial fraction, total mesangial volume and BMT
For electron microscopy, small blocks (2 x 2 x 4 mm) were cut perpendicular to the cortex and embedded in Epon, cut on an Ultramicrotome (Reichert Ultracut S, Leica, Vienna, Austria) and stained with uranyle acetate and lead citrate. In each animal, two blocks were cut for electron microscopy and three glomeruli were examined. Images were recorded with a video camera (Proscan, Münster, Germany) mounted on a Tecnai 12 electron microscope (Phillips, Enthoven, The Netherlands). The mesangial fraction was determined by point counting the image on a monitor at a final magnification of 4400x as previously described [12]. The total mesangial volume was calculated by multiplying the mesangial fraction by the total glomerular volume. For determination of BMT, images at a final magnification of 42 000x were used. The BMT was measured by AnalySIS (Proscan, Münster, Germany) applying orthogonal intercept as previously described and given as a harmonic mean [13].
Statistical analysis
All results are given as mean values±SEM, with n indicating the number of rats studied. Differences between groups were analysed by one-way analysis of variance (ANOVA) in combination with the unpaired or paired Student's t-test when appropriate. When data did not follow a normal distribution or failed the equal variance test, the MannWhitney Rank Sum test was used to analyse differences between groups. Statistics were performed using GraphPad Prism version 3.00 for Windows 95 (GraphPad Software, San Diego CA; www.graphpad.com).
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Results |
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Serum 8-epi PGF2 levels
No significant differences were observed in the serum levels of 8-epi PGF2 in GotoKakizaki rats and Wistar rats (973±243 vs 2053±533 ng/l, respectively, P = 0.2).
BP, UAE and CCr
BP was not different between the groups at 2 and 8 months of age (Table 1). At 14 months, there was a significant difference in BP between GotoKakizaki and Wistar rats due to a reduction in BP in the Wistar rats of 12% compared with the BP at 8 months (P < 0.05). In both groups, systolic BP remained within the normotensive range at all times, ranging from 91 to 142 mmHg. UAE rose progressively over the study period in GotoKakizaki rats, and the rise in UAE was much more pronounced and significantly higher than in Wistar rats (Figure 1). CCr was similar in both groups at the age of 2 months. In Wistar rats, CCr did not change over time (Figure 2). In GotoKakizaki rats, CCr was significantly decreased at the age of 8 months with no further decrease at 14 months. CCr was significantly lower in GotoKakizaki rats than in Wistar rats at 8 and 14 months.
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Discussion |
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GotoKakizaki rats showed an impressive glomerular hypertrophy of 47% compared with Wistar rats at 14 months, in agreement with previous findings [4]. This glomerular hypertrophy is partly due to expansion of the mesangial region, as both the total and the fractional mesangial volume were higher in the GotoKakizaki than in the Wistar rats. Fractional and total mesangial volume were not different between both strains in a previous study [4]. However, given the small sample size (n = 4), it is possible that the study was underpowered to detect a small effect. Recently, mesangial proliferation evaluated by light microscopic semiquantitation of glomerular lesions and smooth muscle actin immunostaining was also higher in male GotoKakizaki rats than in age-matched Wistar rats [6]. The basement membrane thickening observed in the female GotoKakizaki is comparable with that found in studies reported earlier, indicating that basement membrane thickening is age dependent [4,14].
The progressive increase in UAE was much more prominent in GotoKakizaki than in non-diabetic Wistar rats, in contrast to previous studies in mostly male Goto-Kakizaki rats [3,4,6]. We studied female rats because male rats are known to spontaneously develop proteinuria of glomerular origin and glomerulosclerosis [15]. In addition, proteinuria in male rats is characterized by elevated excretion of the sex-dependent 2u-globulin and by increased albumin excretion with increasing age, resulting in alterations in the composition of urinary proteins with age [16,17]. It is therefore important to include only female animals when the influence of diabetes on renal functional changes is investigated. Due to the design of the study and our primary interest in glomerular morphology, we chose to determine the CCr as an estimate of glomerular filtration rate (GFR) instead of the gold-standard procedure, inulin clearance. CCr was significantly lower in GotoKakizaki rats than in Wistar rats at the age of 8 and 14 months, in contrast to previous results [3,4,6]. However, as these studies simply measured serum creatinine levels [35] and/or included only male GotoKakizaki rats [3,5,6], results cannot be compared.
Despite similar renal structural changes, i.e. glomerular hypertrophy, BMT and mesangial expansion, female, but not male, GotoKakizaki rats develop progressive proteinuria and renal failure, suggesting an effect of gender. Generally, males with non-diabetic renal disease progress more rapidly than females, but the effect of sex on the course of diabetic nephropathy remains to be established [18,19].
GotoKakizaki rats had a slightly higher BW than Wistar rats, but FC and total BW gain were similar in both groups. Whether this difference in BW is due to a difference in body composition is unknown. Kidney weight influences glomerular volume but it is unlikely that the small increase in kidney weight observed in GotoKakizaki rats is solely responsible for the prominent increase in glomerular volume. In humans, glomerular volume correlates independently with body surface area and not with kidney weight [20].
GotoKakizaki rats are characterized by hyperglycaemia and glucose intolerance [2,14]. In contrast to other animal models of type 2 diabetes such as the obese Zucker diabetic fatty rat, the OtsukaLongEvansTokushima fatty rat, the ob/ob mouse and the db/db mouse [21], the GotoKakizaki rat lacks obesity, hypertension and dyslipidaemia, factors which may result in renal damage independent of diabetes. The GotoKakizaki rat may be a useful model to study the pathogenesis of diabetic kidney disease in the absence of these confounding variables. In addition, mice models have the disadvantage that information on BP is often not available.
It is interesting to note that the GotoKakizaki rat develops severe renal functional and structural changes in spite of rather mild diabetic metabolic changes. This suggests that, although hyperglycaemia is the most important risk factor for the development of microvascular complications in diabetes [22], other factors are also involved. Serum 8-epi PGF2 levels, a parameter of oxidative stress, were not different between Wistar and GotoKakizaki rats. However, as we did not measure other parameters of oxidative stress, the role of oxidative stress in this animal model remains to be determined. Transient hyperglycaemia may stimulate other factors such as growth factors and cytokines which activate further downstream molecules and pathways that lead to pathological renal events even when glycaemia is normalized again.
In Europe and America, the majority of patients with type 2 diabetes (>80%) are obese using body mass index (BMI) criteria of >25 kg/m2 for women and >27 kg/m2 for men. In developing countries such as India, however, non-obese type 2 diabetic patients constitute a common category (>60%) and many are actually lean with a BMI of <18.5 kg/m2 [23]. Lean type 2 diabetes probably represents a more severe form of diabetes with an increased risk of microvascular complications [23].
In conclusion, the results demonstrate that the female GotoKakizaki rat is a lean, hyperglycaemic, non-hyperinsulinaemic, normotensive experimental model of type 2 diabetes with robust renal structural and functional changes, i.e. glomerular hypertrophy, BMT, mesangial expansion, progressive increase in albuminuria and decline in GFR, that resemble the changes found in long-term type 2 diabetic patients. The GotoKakizaki rat may be a useful model to study the pathogenesis of diabetic nephropathy in lean type 2 diabetes, a condition which is common in developing countries.
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Acknowledgments |
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Conflict of interest statement. None declared.
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References |
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