1 Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin
2 Human and Molecular Genetics Center, Medical College of Wisconsin, Milwaukee, Wisconsin
3 Department of Pathology, University of Dundee, Dundee, Scotland
4 Department of Dermatology, Medical College of Wisconsin, Milwaukee, Wisconsin
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ABSTRACT |
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One of the major morbidity and mortality factors confronted by patients with diabetes is an increased risk of developing diabetic nephropathy that often progresses to end-stage renal disease (ESRD) (13). A long-standing question pertaining to the development of renal disease in diabetes concerns the mechanisms involved in this process. A wealth of data have been generated on possible mechanisms by which diabetes and its ancillary metabolic, hemodynamic, growth, and glomerular cell injury-related alterations may modulate the progression of diabetic nephropathy (47). Nevertheless, the observation that approximately two-thirds of patients with diabetes do not develop renal disease indicates that hyperglycemia is a permissive factor in diabetic nephropathy, but elevated plasma glucose levels alone do not fully account for renal injury (1). Thus, genetic factors are thought to play an important role in the susceptibility for diabetic nephropathy, with several clinical and epidemiologic studies supporting this hypothesis (810).
The complex interplay between diabetes-dependent and -independent factors in determining the progression of renal disease could become more amenable to study if there were adequate animal models that spontaneously develop diabetes and renal lesions that mimic those seen in patients with diabetic nephropathy. However, to date, no rodent model of diabetes that fully recapitulates the chronology of events and histologic changes in the kidney that are characteristic of patients with diabetic nephropathy has been developed. Several rodent models of spontaneous diabetes, such as the Zucker rat, BB rat, and DB mice, exhibit glomerular hypertrophy, thickening of basement membranes, and diffuse expansion of the glomerular mesangial matrix that resemble some of the histologic changes seen in diabetic nephropathy (1114). However, these models, unlike human diabetic nephropathy, do not exhibit progressive expansion of mesangial matrix leading initially to focal glomerulosclerosis and proteinuria, continuing with the development of severe diffuse, global glomerulosclerosis with nodule formation and ESRD, characterized by elevations in plasma urea and creatinine levels and decreased GFR.
One strain of a spontaneously diabetic rat that may harbor genetic factors predisposing them to renal disease is the GK rat, a nonobese, normotensive model of non-insulin-dependent diabetes (type 2 diabetes). GK rats display glucose intolerance as early as 2 weeks of age (high basal plasma insulin levels) and exhibit elevated plasma glucose levels after the administration of a glucose load by 4 weeks of age (1517). By 12 weeks of age, GK rats exhibit frank type 2 diabetes characterized by elevated fasting glucose and insulin levels and a prolonged elevation in plasma glucose levels after an oral glucose load.
Several studies have reported that GK rats also exhibit common histologic changes in the kidney seen in other animal models of diabetes, including thickening of the glomerular basement membranes, mild mesangial matrix expansion, and glomerular hypertrophy (18,19). Nevertheless, follow-up studies indicated that even old GK rats do not exhibit progressive renal disease characterized by the development of global glomerulosclerosis and nodule formation, marked proteinuria, and ESRD (20,21). Thus, a new animal model of diabetic nephropathy is sorely needed to study the genetic basis of this disease, to identify biomarkers and diagnostic tests for susceptibility to diabetes-related complications, and to develop new therapeutic targets and strategies that might alter the progression of diabetes and diabetic nephropathy. In this study, we report on the characterization of a new rat model that develops type 2 diabetes and progressive diabetic nephropathy, leading to ESRD.
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RESEARCH DESIGN AND METHODS |
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Genetic comparison of the T2DN and GKFL rats.
For determining the degree of genetic relatedness between GK and T2DN rats, a second colony of GK rats, termed GKFL (purchased from Dr. Robert Farese, Tampa, FL), had to be used, given our inability to obtain more animals from the Swedish colony. A genome-wide scan with 681 SSLPs distributed across the genome was carried out to assess the degree of the FHH genome retained in the T2DN strain, as well as differences between T2DN and GKFL rats. The SSLPs selected were polymorphic between GK and FHH rats and exhibited a high degree of polymorphism among the other 47 rat strains characterized in our previous studies (22), maximizing the likelihood of detection of polymorphisms between the GK strains (rgd.mcw.edu).
Genotyping.
DNA was extracted from a 1-mm3 section of tail that was incubated in 500 µl of lysis buffer overnight at 55°C, followed by an isopropanol precipitation and resuspension in TE buffer. DNA was diluted to a final concentration of 5 ng/µl. The rats were genotyped using PCR. Before PCR, the primers were radiolabeled with 32P--ATP. PCR was carried out, and the products were electrophoretically separated in 6% polyacrylamide gels, as previously described (22).
Characterization of diabetes and glucose intolerance.
Male BN, FHH, T2DN, and GKFL rats were subjected to an intraperitoneal glucose tolerance test (IPGTT) at 3, 6, 9, and 12 months of age. Before the test, all animals were subjected to two to three training sessions. After the determination of fasting (12 h) glucose levels, the animals received an injection of 1 g/kg of a 2.8-mol/l glucose solution, intraperitoneally. Approximately 10-µl blood samples were collected from an incision in the tail at 30, 60, 90, and 120 min after the glucose load. Plasma glucose levels were measured using reagent strips read in a glucose meter (Bayer, Elkhart, IN).
Proteinuria and albuminuria.
Male BN, FHH, T2DN, and GKFL rats were placed in metabolic cages at 1, 3, 6, 9, 12, and 18 months of age, and urine was collected for 24 h. Total protein concentration in the urine was determined colorimetrically using the Bradford method (BioRad, Hercules, CA) (23). Albumin was measured using the albumin blue 580-fluorescence assay (24).
Creatinine.
Serum creatinine levels were determined at 12 and 18 months in male T2DN and GKFL rats (22 months as well for this strain), using a modification of the Jaffe color reaction.
Determination of lipid profiles.
Serum cholesterol and triglyceride concentrations were measured in BN, T2DN, and GKFL rats at 6 and 12 months of age. For this procedure, the rats fasted overnight, and 500700 µl of blood was collected from the tail vein. Total cholesterol and triglycerides were determined using kits from Sigma Diagnostics (St. Louis, MO).
Histology.
Renal histology was assessed in T2DN and GKFL rats at 6, 12, 18, and 22 (GKFL only) months of age. The right kidney was removed, weighed, and then fixed in 10% formalin solution followed by imbedding in paraffin. Two 4-µm-thick sections were prepared and stained with periodic acid-Schiff. The sections were examined by light microscopy for the degree of vascular injury, renal interstitial fibrosis, and the degree of glomerulosclerosis and expansion of the mesangial matrix in the glomerulus. Lesions in individual glomeruli were scored on a 0 to 4+ scale with 0 representing a normal glomerulus, 1+ representing a 125% of loss of capillaries in the tuft, 2+ representing a 2650% loss, 3+ representing a 5175% loss, and 4+ representing >75% of the tuft sclerosed. A total of 3035 glomeruli per kidney were analyzed, and an average score (sclerosis index) was calculated for each animal. Glomerular diameter and volume were also determined using a modification of the Maximal Planar Area method (25).
For electron microscopy, kidneys were removed and placed in cold 3% glutaraldehyde fixative in 0.1 mol/l cacodylate buffer (pH 7.4). Tissue was cut in 1-mm3 blocks and further fixed overnight at 4°C. After postfixation, tissues were embedded in Epon/Araldite, and sections were cut from epoxy blocks. Sections were mounted on 200-mesh copper grids and observed and photographed at 11,000x.
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RESULTS |
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Genomic comparison of T2DN and GKFL rats.
A genome-wide scan with 543 polymorphic markers from the 681-marker set was carried out for comparison between T2DN and GKFL rats. The results indicate that the two strains of GK rats are 98% identical (535 of 543 markers). Of the eight markers that were different (these are not FHH alleles but alleles that differ between the GK substrains from Sweden and Florida), three were on independent chromosomes: 3 (D3Rat157), 11 (D11Mgh5), and 12 (D12Rat22). Five additional polymorphic markers were identified on chromosome 1: D1Rat291, D1Mit18, D1Mit34, and D1Mgh12 within 30 cM from each other. The fifth difference at D1Rat185 mapped 57 cM from the telomere on chromosome 1 (Fig. 1). Together with the genomic segments carried over from the FHH rats, the genotyping results indicate that T2DN and GKFL strains are 97% identical at the microsatellite level across the entire genome.
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Development of diabetes and glucose intolerance.
Baseline fasting glucose levels were elevated to >200 mg/dl by 3 months of age in both T2DN and GKFL rats and were significantly greater than the values seen in nondiabetic BN and FHH rats (Fig. 2). However, no significant difference in fasting glucose levels was seen between GKFL and T2DN rats at any point during the study. Both GKFL and T2DN exhibited glucose intolerance as indicated by the increased area of the plasma glucose clearance curve after an IPGTT. The degree of glucose intolerance was greater in T2DN than in GKFL at 3 months of age, but no significant difference was observed in 6- and 9-month-old rats. After 12 months of age, the degree of glucose intolerance in T2DN rats was 2030% greater than that seen in age-matched GKFL rats.
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To determine whether T2DN rats develop progressive renal disease leading to ESRD, we measured plasma creatinine concentrations at 12 and 18 months in BN, GKFL, and T2DN rats. Serum creatinine concentration did not increase in GKFL rats compared with age-matched BN rats (Fig. 3), indicating that GKFL rats do not exhibit progressive renal disease leading to ESRD. In contrast, in T2DN rats, serum creatinine levels rose from 0.58 ± 0.01 at 12 months to 1.66 ± 0.26 mg/dl at 18 months (five times the normal level for a rat), indicating that T2DN rats exhibit a progressive decline in renal function.
Dislipidemia.
Given the strong correlation between proteinuria and dyslipidemia in human diabetic nephropathy (33,34), we compared serum cholesterol and triglyceride levels in T2DN and GKFL rats (Table 1). Serum cholesterol levels in 12-month-old male T2DN rats averaged 170.4 ± 14.0 mg/dl and are four times higher than the levels measured in age-matched male BN rats (41.7 ± 1.4 mg/dl). Serum triglyceride concentration was also elevated in 12-month-old T2DN rats (157.6 ± 23.8 mg/dl) compared with the values seen in age-matched control BN rats (34.0 ± 5.1 mg/dl). In 12-month-old male GKFL rats, both serum triglyceride (108 ± 3 mg/dl) and cholesterol levels (66 ± 6 mg/dl) are elevated as compared with BN rats but are still significantly lower than the corresponding values obtained in age-matched T2DN rats. Proteinuria and dyslipidemia are strongly correlated in T2DN rats with a correlation coefficient between proteinuria and serum cholesterol levels of 0.80 (P < 0.01) and that between proteinuria and serum triglycerides levels of 0.77 (P < 0.01) at 12 months.
Histologic changes in the kidney.
Histologic analysis of the kidneys of T2DN rats revealed an extensive pattern of progressive renal disease characterized by glomerular, tubular, and renal interstitial injury. As illustrated in Fig. 4, the predominant form of glomerular alteration at 12 months of age in T2DN rats is glomerular hypertrophy (Fig. 4B) and focal segmental glomerulosclerosis, with regional adhesion of the glomerular tuft to Bowmans capsule associated with expansion of the mesangial matrix and filling in of capillaries (Figs. 4C and D), is more severe than that seen in age-matched BN (Fig. 4A) and GKFL rats (Fig. 4E). Electron microscopy reveals a similar pronounced thickening of both glomerular and tubular basement membranes in the kidneys of both GKFL and T2DN diabetic rats (Figs. 4G and H) relative to BN rats (Fig. 4F).
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DISCUSSION |
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In the present study, we demonstrated that T2DN rats are genetically identical to GKFL rats at 97% of the 681 SSLP markers tested across the genome. Of the 3% differences between the strains, only 1% is accounted for by passenger loci from the FHH genome retained in T2DN rats. None of these passenger loci overlap with genomic regions corresponding to quantitative trait loci (QTLs) reported to be modulating the development of proteinuria, renal morphologic abnormalities, or hypertension in FHH rats (30,37). Moreover, the renal histopathologic findings and time course of renal disease in T2DN rats are very different from those observed in FHH rats (3032,37). Together, these data suggest that diabetic nephropathy in T2DN rats is altogether a distinct pathological entity from the renal disease of FHH rats. We conclude that the differences in the development of diabetic nephropathy in T2DN and GKFL rats are likely due to small differences in their genomic background and/or their different mitochondria.
There is a major difference between T2DN and GKFL rats on chromosome 1, with five polymorphisms clustering around a 57-cM genomic segment. These polymorphisms, in an otherwise isogenic background, suggest that the last common progenitor of T2DN and GKFL rats carried different haplotypes in this genomic segment and that each GK strain eventually retained one allelic variant. This finding is of special importance in the light of previous studies that revealed a major QTL, termed Niddm1, mapping to this same genomic segment on chromosome 1, linked to diabetes in GK rats (25,28). This QTL was later confirmed in congenic strains to be a major factor determining hyperglycemia in GK rats (29), but its role in determining the development of renal disease is unknown. Future studies aiming at dissecting the genetic basis of renal disease in T2DN rats will reveal whether similar genes are responsible for their susceptibility to diabetes and renal disease. Given the genetic similarities between GKFL and T2DN rats and that both strains develop type 2 diabetes, it is feasible to envision a scenario in which the added susceptibility to progressive renal disease in T2DN rats is due to genetic factors independent from diabetes, that were exclusively captured in the T2DN strain.
Glomerular hypertrophy is evident in both T2DN and GKFL rats several months before the development of overt renal disease in T2DN rats. These findings seem to corroborate earlier reports that GK rats exhibit glomerular hypertrophy and thickening of basement membranes that is commonly reported in most experimental models of diabetes (1821). Unlike these other models, the natural course of renal disease in T2DN rats closely parallels that of human diabetic nephropathy. Renal structural abnormalities such as glomerular and tubular hypertrophy are already observed at early ages and precede the development of proteinuria. The most common presentations of glomerular damage in T2DN rats at 12 months of age are severe focal segmental glomerulosclerosis and expansion of the mesangial matrix, with obliteration of glomerular capillaries and the formation of nodular lesions in several glomeruli. By 18 months of age, there is further expansion of the mesangial matrix, leading to diffuse global glomerulosclerosis with the formation of many large, acellular nodules in many glomeruli. The presence of nodular glomerulosclerosis, clearly discernible in T2DN rats with long-standing type 2 diabetes, is a pattern consistent with the development of diabetic nephropathy in humans (5,36). GKFL rats did not develop severe focal glomerulosclerosis or nodules even at 22 months of age, in the face of lifelong diabetes. Thus, the T2DN rat represents the first reported rodent model of spontaneous diabetes that develops progressive renal disease with the formation of nodular glomerulosclerosis.
Both T2DN and GKFL develop mild dyslipidemia, as reflected by elevated levels of plasma cholesterol and triglycerides. In T2DN rats, proteinuria and dyslipidemia are strongly correlated. This is consistent with previous reports in diabetic patients that demonstrate a strong correlation between dyslipidemia and progression of diabetic nephropathy (33,34). It is interesting that GKFL rats display a milder form of dyslipidemia. This likely reflects the milder proteinuria and lack of renal disease observed in these rats. These data seem to support the notion that the loss of plasma proteins triggers abnormalities in lipid metabolism perhaps as a result of loss of protein binding and explains the close association between proteinuria and lipid abnormalities in most forms of ESRD (3840). Nevertheless, there is mounting evidence that alterations in lipid metabolism may also contribute to the renal damage seen in diabetes (41,42).
In summary, the present study characterized the first rodent model of spontaneous type 2 diabetes (T2DN rats) that develops progressive proteinuria and glomerulosclerosis leading to formation of nodules and ESRD. It also identified a closely related control strain of GK rats (GKFL) that develops diabetes but is resistant to the development of progressive proteinuria and renal disease. Discrete and yet unidentified genetic differences between these two strains of rats likely determine the difference in the susceptibility to develop diabetic nephropathy between T2DN and GKFL rats. These small differences, with large phenotypic impact, make these two strains ideal models for genetic dissection of diabetes-associated renal disease, as well as the relationships between the duration and severity of diabetes and the later onset and progression of renal disease.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address correspondencereprint requests to Howard J. Jacob, PhD, HumanMolecular Genetics Center, 8701 Watertown Plank Rd., Medical College of Wisconsin, Milwaukee, WI 53226. E-mail: Jacob{at}mcw.edu
Received for publication July 17, 2003 and accepted in revised form December 1, 2003
ESRD, end-stage renal disease; IPGTT, intraperitoneal glucose tolerance test; QTL, quantitative trait locus; SSLP, simple sequence-length polymorphism
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REFERENCES |
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