Evolution of Metabolic and Renal Changes in the ZDF/Drt-Fa Rat Model of Type II Diabetes1’2

Evolution of Metabolic and Renal Changes in the ZDF/Drt-fa Rat Model of Type II Diabetes1’2

Jiten P. Vora, Stephanie M. Zimsen, Donald C. Houghton, and Sharon Anderson3

utility of this model for study of diabetic renal disease

J.P. Vora, SM. Zimsen, S. Anderson, Division of Nephrol- Is compromised by the ubiquitous presence of other, ogy, Hypertension, and Clinical Pharmacology, De- nondlabetic renal lesions. partment of Medicine, Oregon Health Sciences Uni- Key Words: Albuminuria. hyperinsulinemia. pyelonophritis. hy- versity, Portland. OR, and Portland Veterans Administration Medical Center, Portland, OR dronephrosis. hyperilpidemia D.C. Houghton. Division of Anatomical Pathology. De- U nderstanding of the pathophysiobogy of renal partment of Pathology, Oregon Health Sciences Uni- disease In non-insulin- dependent diabetes mel- versity, Portland, OR litus (NIDDM) has been hampered by the lack of appropriate animal models of the disease. Over a (J. Am. Soc. Nephrol. 1996; 7:113-117) dozen putative models have been reported (1), but many are not truly representative of the human dis- ABSTRACT ease. For example, some models exhibit only mild hyperglycemia, whereas others do not develop any Studies of the pathophysiology of renal disease In renal manifestations. In most of these models, evolu- non-insulin-dependent diabetes mellitus (NIDDM) tion of diabetic nephropathic changes has not been have been hindered by the lack of an appropriate examIned (1). experimental model. We examined the natural history The partially Inbred Zucker Diabetic Fatty Rat of metabolic and renal changes in the partially in- (ZDF/Drt-fa) is a relatively new model that more bred Zucker Diabetic Falfy Rat (ZDF/Drt-fa), a model closely mimics the metabolic status of human MDDM that closely mimics the metabolic abnormalities of (2,3). DerIved from a few Zucker rats In which unusu- NIDDM. Lean nondiabetic fl#ermates served as con- ally high blood glucose levels developed, this strain trols. Body weights in the obese rats were higher has been selectively inbred for more than 16 genera- initially, but thereafter stabilized at values similar to tions (3). Affected obese males routinely develop stable those in lean controls. Blood glucose levels rose to hyperglycemia, and have been reported to exhibit diabetic neuropathy (4). Serum insulin levels are ini- overtly hyperglycemic levels in the obese group, tially high, but then decline as pancreatic 13cells cease stabilizing in the 300 to 400 mg/dL range. Serum to respond to the glucose stimulus (5). Accordingly, we insulin, cholesterol, and triglyceride levels were all sought to determine whether this strain might provide elevated in the obese group, though insulin levels an appropriate model of diabetic nephropathy. declined later in life. Values for systolic blood pressure rose slightly with age in both groups, but remained METHODS within the normal range, and did not differ between Longitudinal studies were performed in 20 obese male groups. Urinary albumin excretion values were higher ZDF/Drt-fa rats, obtained at the age of 5 wk from Genetic In the obese group at all time points, and rose pro- Models, Inc. (Indianapolis, IN). An addItional 20 male lean littermates served as controls. When unexpected changes gressively throughout the study. Morphologic exami- were found in rats followed to 40 wk of age (discussed below), nation revealed the presence of severe hydronephro- additional groups were obtained and followed to 12 (N = 9 sis in almost all animals, affecting lean as well as each) and 16 (N = 7 to 9) wk of age, to determine the timing obese rats. In some cases, complications were found, and onset of these abnormalities. All rats were fed tap water including tubular dilation, necrotizing granulomas, and Purina Rodent Chow 5008 (Ralston Purina, Richmond, inflammatory changes, and pyelonephritis, some of IN), containIng 6.5% fat, ad llbttum. The experimental proto- cols were approved by the Institutional Animal Care and Use which were fungal. Accordingly, the ZDF/Drt-fa rat Conunittee. appears to be an excellent model of the metabolic Blood glucose (BG) levels in tail venous blood were moni- changes that characterize NIDDM. Unfortunately, the tored weekly until the development of hyperglycemia, and then monthly thereafter. Monthly measurements were also

1 Received March 13, 1995. Accepted August 29. 1995. made of systolic blood pressure (SBP) by the conscious 2Poons of these studies were presented at the 1993 Annual Meeting of the tail-cuff method, body weight. and 24-h urinary albumin American Society of Nephrology, and published In abstract form (J Am Soc excretion value. Animals were followed until 40 wk of age. at Nephroi 1993;4:807). which time they were anesthetized with mactin (Research 3correspondence to Dr. S. Anderson, Div. of Nephroiogy and Hypertension Biochernicahs, Natick, MA; 100 mg/kg ip). Blood was taken in PP262, Oregon Health Sciences UniversIty, 3314 SW. U.S. Veterans Hospital Rd., non-fasting conditions for determination of serum glycosy- Portland, OR 97201. lated hemoglobin, insulin, cholesterol, and triglyceride lev- 10466673/0701-01 13103.00/0 Journal of the American Society of Nephrology els, after which the kidneys were fixed by perfusion for 5 mm Copyright © 1996 by the American Society of Nephroiogy at a pressure of 120mm Hg wIth 1.25% glutaraldehyde in 0.1

Journal of the American Society of Nephrology 113 Natural History of the ZDF/Drt-fa Diabetic Model

M sodium cacodylate buffer (pH 7.4). After perfusion-fixa- tion, kidneys were immersed In the same fixative for 24 h and 600A then rinsed in Tyrodes solution for another 24 h. A series of 3 mm-thick transverse sections from each kidney were pro- 400 cessed and embedded in paraffin for light microscopy. Two to 4 micron-thIck sections were stained with periodic acid- 200 0 Schiff stain and examined by light microscopy. Incidence of hydronephrosis, tubular dilation, and focal and segmental glomerular sclerosis (FSGS) were assessed semiquantita- 500 B * tively. by using a score ranging from 0 (no lesions) to 3 .. 400 (extensive lesions). Specifically, the grading scale used was: 0 300 = absent/normal; 1 = minimal, few instances seen through- , 200 out the sections; 2 = moderate, instances easily found, most 10 areas normal; 3 = extensive, instances found In nearly all 400X fields. For hydronephrosis, the following criteria were used: 0 = normal (finger-in-glove configuration of the papilla 160 C and calyx); 1 = minimal (a narrow but definable fluid-ifiled 120 calyceal space with normal papillary contour): 2 = moderate (unequivocal dilatation of the calyx. with compression of the 80 papifia. but with preservation of its conical shape); 3 = a- w 40 marked (gross distension of the calyx, typically increasing (I, overall volume of the kidney by at least 50%. and resulting in 0 severe compression of the lateral cortex and distortion of the * 500 D papifia). ‘S Urinary albumin concentration was determined using a specific ELISA assay (Nephrat. Exocell, Inc., Philadelphia, 1 300 PA). Glycosylated hemoglobin was determined by afilnity w 200 * * column chromatography (Glyco-Gel B, Pierce Chemical Co., :: 100 Rockford, IL). Serum cholesterol and triglyceride levels were measured by enzymatic colorimetric methods (Cholesterol 6- * and Triglyceride GPO-Trlnder kits, Sigma Diagnostics. St. 4- Louis, MO) by using a centrifugal analyzer (Cobas#{174}-Bio. Roche Analytical Instruments. Inc., Nutley. NJ). Serum in- 2- sulin levels were measured by radioimmunoassay (Rat Insu- 0 lin RIA Kit, Linco Research, Inc., St. Louis, MO). Results are 6812162024283236 presented as means ± SE. Comparisons between lean and Age (weeks) obese groups were performed by repeated measures analysis of variance, followed by Tukey’s comparisons or the Mann- Figure 1. Serial measurements of (A) body weight. (B) blood Whitney test, as appropriate. Statistical significance was glucose. (C) systolic blood pressure (SBP), and (D) 24-h defined as P < 0.05. urinary albumin excretion (UAE), in nondiabetic lean (D) and diabetic obese () rats. Note the break in the scale in

RESULTS Figure 1D. Values are means ± SE. * P < 0.05 versus lean at same time point. By 6 wk of age, the characteristic changes in body habitus that occur in obese rat strains were already apparent, and body weight was higher in the obese triglyceride (410 ± 43 versus 33 ± 2 mg/dL, P < group (lean, 138 ± 4 g; obese. 177 ± 5 g; each N 10: 0.00 1) levels, as compared with lean rats. By 36 wk of P < 0.01). Blood glucose levels (lean, 77 ± 3 mg/dL; age, serum cholesterol (423 ± 28 versus 106 ± 3 obese 82 ± 3 mg/dL; each N = 10; P>0.05) confirmed mg/dL, P < 0.00 1) and triglyceride (969 ± 157 versus that hyperglycemia was not yet present. Subsequent 74 ± 12 mg/dL, P < 0.00 1)levels remained elevated in changes in body weight, blood glucose level, systolic the obese diabetic rats. Though hyperglycemia was blood pressure, and albuminuria are summarized in sustaIned in the obese group, serum insulin levels Figure 1. Body weights remained higher in the obese were no longer different from those in lean control rats diabetic group during the first six months of age, but (48 ± 3 obese versus 96 ± 14 .tU/mL, NS). Systolic thereafter both groups were stable and body weights blood pressures were within the normal range in both converged (Figure 1A). Blood glucose levels were com- groups throughout the time course. They rose slightly parable in the two groups of rats at 8 wk of age. but with aging In both groups, but did not differ between thereafter uniformly rose to overtly hyperglycemic lev- groups (Figure 1C). Values for 24-h urinary albumin els in the obese diabetic group (Figure 1B). Other excretion in obese diabetic rats were slightly, though metabolic characteristics of NIDDM were apparent, as significantly, higher than those in the lean rats at 6 wk well. By 12 wk of age. obese diabetic rats exhibited of age (1.4 ± 0.4 versus 0.5 ± 0.1 mg/day, P < 0.05). significant elevations of serum insulin (256 ± 28 Thereafter, albuminuria in the obese diabetic group versus 80 ± 9 jU/mL, P < 0.001), serum cholesterol rose progressively (Figure 1 D), reaching 418 ± 57 (182 ± 5 versus 82 ± 3 mg/dL, P < 0.001), and serum (range, 107 to 761) mg/d by 36 wk. By comparison,

114 Volume 7 - Number 1 1996 Vora et a)

values in the lean rats rose only slightly (Figure 1D), so in five animals. These tissues were not evaluated that values were significantly higher in the obese systematically to determine the responsible microor- group at all time poInts. ganism. Fungi were found in the tissues of three Renal morphologic changes at 40 wk of age are animals showing granulomatous inflammation (Fig- summarized in Table 1, and Figures 2 and 3. When ure 2). Thus, nondiabetic renal lesions (arising as the severe incidence of hydronephrosis at 40 wk of age complications of hydronephrosis) were prominent in was recognized, additIonal groups were obtained and this strain, occurring in lean rats but more promi- followed to 12 and 16 wk of age. By 12 wk of age, obese nently in the diabetic rats. diabetic rats were slightly heavier and already exhibited renal hypertrophy, both in absolute terms and DISCUSSION when expressed as a percentage of body weight. Chronic hyperglycemia was confirmed by elevation of During the past decade, substantial progress has HBASC values. Hydronephrosis was grossly apparent been made toward understanding of the pathogenesis In some of the lean rats, and most of the obese rats, at of nephropathy that occurs as a result of 1’ype I this early time point. At all time points, the hydrone- (insulin-dependent) diabetes. Experimental (6,7) as phrosis was found in both lean and obese rats. It was well as clinical (8) studies have indicated rational often bilateral, occurred at highly variable locations in therapeutic regimens that hold promise in delaying or the ureter, and was not associated with any discern- preventing renal complications. In contrast, the thle anatomic obstruction. At 12 wk of age, the hydro- mechanisms that underlie nephropathy associated nephrosis scores did not dIffer between obese and lean with NIDDM are much less well defined. Difficulties rats. However, other lesions were occasionally found arise, in part, because of the heterogeneous nature of in the obese rats, with one rat showing papillary the disease, and the unclear natural history of the calcifications, one showing focal tubular dysplasia, renal manifestations. However, understanding has and one showing minimal focal scarring. Interestingly, also been hindered by lack of appropriate experimen- tubular dilation was already more prominent in the tal models. The recently described partially inbred diabetic group. Tubular dilation consisted of marked ZDF/Drt-fa rat appears to circumvent many of the distension of individual tubular segments, containing defects in other models. The present findings confirm colloid-like cast material. This was most extensive in previous reports (3,4,9) that hyperglycemia is sub- tubule segments near the corticomedullary junction, stantial in magnitude, uniform in occurrence, and but was found at every level of the cortex. By 16 wk of sustained over the long term in affected males of this age, absolute and relative renal hypertrophy was more strain. We also found that as in clinical NIDDM, and prominent in the diabetic group, as was the incidence as previously noted in thIs straIn (9). serum insulin of hydronephrosis. One diabetic animal was noted to levels evolve from hyperinsulinemic to hypoinsuline- have confluent necrotizing granulomas, uroepithelial mic, whereas serum cholesterol and triglyceride levels hyperplasia, interstitial fibrosis, and chronic inflam- are elevated. Thus, the metabolic profile In this model matory changes (Figure 2). By 40 wk of age, diabetic appears to be consonant with that In clinical NIDDM, rats showed continued renal hypertrophy and hyper- and the model should prove very useful in studies glycemia. The incidence and severity of hydronephro- related to metabolically relevant interventions. The sis (Figure 3), tubular dilation, and focal and segrnen- absence of systemic hypertension is also useful, as tal glomerular sclerosis were all significantly higher in confounding Issues of differences In blood pressure the diabetic group; only one diabetic rat was free of are not a problem. hydronephrotic changes. In addition, evidence of Another finding of potential relevance is the pres- granulomatous and/or infectious complications was ence of renal hypertrophy in this model. Though well- more apparent, with various lesions being prominent documented in humans and animals with Type I

TABLE 1. Histologic studies at 12, 16, and 40 wk#{176}

Hydro. Tub. DII. FSGS TKW/100 Group ( BW (g) 1KW (g) HBAIC (%) (mean (mean (mean BW / score) score) score)

Lean-12 wk (9) 364 ± 4 2.49 ± 0.06 0.68 ± 0.02 2.4 ± 0.4 0.8 ± 0.2 0.0 ± 0.0 0.1 ± 0,1 Obese-12 wk (9) 388 ± 8’ 3.14 ± 0.06#{176} 0.81 ± 0.01#{176} 10.7 ± 0.40 1.1 ± 0.3 0.4 ± 0,2’ 0.1 ± 0.1 Lean-16 wk (]0)b 387 ± 8 3.04 ± 0.10 0.80 ± 0.01 5.5 ± 0.2 0.2 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 Obese-b wk (9)C 440 ± 4’ 5.09 ± 0.25#{176} 1.16 ± 0.07#{176} 15.7 ± 05#{176} 1.2 ± 0.2’ 0.6 ± 0.3 0.6 ± 0.3 Lean- 40 wk (20)d 520 ± 4 3.31 ± 0.06 0.64 ± 0.02 4.6 ± 0.3 0.8 ± 0.2 0.0 ± 0.0 0.0 ± 0.0 Obese-40 wk (20)#{176} 493 ± 10’ 6.02 ± 0.29#{176} 1.20 ± 0,07g 16.1 ± 1.0#{176} 1.5 ± 0.2#{176} 1.5 ± 0.2#{176} 1.9 ± 0.20

#{176} are means ± SE. BW, body weight; TKW, total (left + right) kidney weight; HBA1C, glycosylated hemoglobin; Hydro., hydronephroSis; Tub. Dii.. tubular dilation; FSGS, focal and segmental giomerulosclerosls. “P < 0.05, ‘P < 0.02; #{176}P < 0.001 versus corresponding lean group. For morphologic studies, ‘ N = 9; C N = 7; d N = 18; e N = 19. Versus corresponding lean group, ‘P < 0.02; #{176}P < 0.001; “P < 0.05.

Journal of the American Society of Nephroiogy 115 Natural History of the ZDF/Drt-fa Diabetic Model

Figure 2. This section shows a margin of the papilla and calyceal fornix from the kidney of an obese rat, at 16 wk of age, showing granulomatous pyelonephritis. The inflammatory infiltrate, which extends broadly into the medulla from the calyx (middle, left). Is densely fibrotic and contains many giant cells. The calyx contains blood and inflammatory exudate (57x; H&E).

stones was found. This fact, together with its presence at highly variable levels along the ureter, suggest a neurogenic origin: alternatively, anatomic distur- bances related to relative ureteral redundancy or tor- tuosity may be responsible. Whatever its etiology, hydronephrosis was found in both obese and lean animals, suggesting that genetics (rather than dis- placement because of fat-pad growth, polyuria as a result of hyperglycemia, or other problems specific to the obese diabetic state) was a primary factor in its development. The hydronephrosis was sufficiently severe as to be grossly apparent, and to result in serious complications including renal abscesses, pyelonephritis, and necrotic granulomas. Finally, we noted that had histologic studies not been performed, the presence of rising albuminuria might have led to the erroneous conclusion that this Figure 3. (A) Transverse section through the kidney of a lean was, Indeed, a model of diabetic nephropathy. These rat,at 40 wk of age, showing no evidence of hydronephrosls observations should reinforce the concept that all (9x; H&E). (B) Transverse section through the kidney of an diabetic albumInuria is not diabetic renal disease, and obese rat, at 40 wk of age, showing marked dilation of the caution should be used in the Interpretation of pub- renal pelvis and compression of the renal paplila. This was lished animal studies that fail to include morphologic graded as 2-3+ hydronephrosis (9x; H&E) confirmation that increased urinary albumin excretion is, indeed, primarily of glomerular origIn, and diabetes, the incidence and importance of renal hy- indicative of pure diabetic nephropathy. pertrophy In Type II diabetes Is less well established. The present studies document the presence of hyper- ACKNOWLEDGMENTS trophy in this model but, unfortunately, the presence These studies were supported, in part, by grants from the Juvenile of hydronephrosls renders this finding more difficult Diabetes Foundation, the American Heart Association (Oregon Aillil- to interpret, given the added water weight. Indeed, the atel, Welcome Trust, Elizabeth Tuckerman Foundation, a VA Re- utility of this model for the study of most aspects of search Advisory Group Award, and the NIH (1RO1 DX 43601, and the diabetic nephropathy is severely compromised by the Clinical Nutrition Research Unit, P 30 DX 40566). J.P.V. was a Fuibright Senior Research Scholar and the recipient of a postdoctoral unanticipated, and unfortunate, occurrence of severe Research Fellowship Award from the Juvenile Diabetes Foundation. hydronephrosis. The lesion does not appear to be S.A. was the recipient of a Career Development Award from the anatomic in nature, as no evidence of strictures or Juvenile Diabetes Foundation. We are grateful to Jean-Baptiste Roul-

116 Volume 7#{149}Number 1 - 1996 Vora et ai

let. Ph.D.. for performing measurements of serum cholesterol, triglyc- down-regulation of 13-cell glucose transporters in non- eride, and insulin levels. insulin-dependent diabetes mellitus may be the cause of diabetic hyperglycemia. Proc Nati Acad Sc! USA 1990;87: REFERENCES 9953-9957. 6. Zatz R, Dunn BR, Meyer TW, Anderson S, Rennke HG, 1. Velasquez MT. Kimmel PL, Michaeiis OE IV: Animal Brenner BM: Prevention of diabetic glomerulopathy by models of spontaneous diabetic kidney disease. FASEB J pharmacological amelioration of glomerular capillary hy- 1990:4:2850-2859. pertension. J Chin Invest 1986:77:1925-1930. 2. Clark JB, Palmer CJ, Shaw WN: The diabetic Zucker 7. Anderson S. Rennke HG, Garcia DL, Brenner BM: Short fatty rat. Proc Soc Exp Biol Med 1983:173:68-75. and long term effects 3. Peterson RG, Shaw WN, Neel M-A, Little LA, Eichberg J: of antihypertensive therapy in the diabetic rat. Kidney Zucker diabetic fatty rat as a model for non-insulin- Int 1988;36:526-536. dependent diabetes meffitus. ILAR News 1990:32:16-19. 8. Lewis EJ, Hunsicker LG, Sam RP. Rohde RD. for the 4. Peterson RG. Ned M-A, Little LA, Kincald JC, Eichberg Collaborative Study Group: The effect of angiotensin- J. Neuropathic complications in the Zucker diabetic fatty converting enzyme inhibition on diabetic nephropathy. N rat (ZDF/Drt-Fa). In: Shafrir E, Ed. Frontiers in Diabetes EngI J Med 1993;329:1456-1462. Research: Lessons from Animal Diabetes, III. London: 9. Bue JM, Shaw WN: ZDR/Drt rat: A new NIDDM model Smith-Gordon: 1990:456-458. with marked susceptibifity to dietary manipulation LAb- 5. Orci L, Ravazzola M, Baetens D, et aL: Evidence that stracti. Diabetes 1991;40(Suppl 1J:217A.

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