Peroxisome Proliferator-Activated Receptor- Agonist

Peroxisome Proliferator-Activated ReceptorAgonist, Rosiglitazone, Protects Against Nephropathy and Pancreatic Islet Abnormalities in Zucker Fatty Rats Robin E. Buckingham, Kamal A. Al-Barazanji, C.D. Nigel Toseland, Mark Slaughter, Susan C. Connor, Andrew West, Brian Bond, Nicholas C. Turner, and John C. Clapham

Rosiglitazone (BRL 49653), a peroxisome proliferator–activated receptor- (PPAR- ) agonist and potent insulin action–enhancing agent, was given in the diet (50 µmol/kg of diet) to male Zucker rats ages 6–7 weeks for 9 months (prevention group). In this treatment mode, rosiglitazone prolonged the time to onset of proteinuria from 3 to 6 months and markedly reduced the rate of its subsequent progression. Progression was also retarded when treatment was commenced (intervention group) after proteinuria had become established (4 months; ages 24–25 weeks). In either treatment mode, rosiglitazone normalized urinary N-acetyl- - D-glucosaminidase activity, a marker for renal proximal tubular damage, and ameliorated the rise in systolic blood pressure that occurred coincidentally with the development of proteinuria in Zucker fatty control rats. The renal protective action of rosiglitazone was verified morphologically. Thus in the prevention group there was an absence of the various indexes of chronic nephropathy that were prominent in the Zucker fatty control group, namely, glomerulosclerosis, dilated tubules containing proteinaceous casts, a loss of functional microvilli on the tubular epithelium, and varying degrees of chronic interstitial nephritis. An intermediate pathology was observed in the intervention group. Also, pancreatic islet hyperplasia, ultrastructural evidence of -cell work hypertrophy, and derangement of -cell distribution within the islet were prominent features of Zucker fatty control rats, but these adaptive changes were ameliorated (intervention group) or prevented (prevention group) by rosiglitazone treatment. These data demonstrate that treatment of Zucker fatty rats with rosiglitazone produced substantial protection over a prolonged period against the development and progression of renal injury and the adaptive changes to pancreatic islet morphology caused by sustained hyperinsulinemia. Diabetes 47:1326–1334, 1998 From SmithKline Beecham Pharmaceuticals (R.E.B., K.A.A., S.C.C., A.W., B.B., N.C.T., J.C.C.), Harlow, Essex and SmithKline Beecham Pharmaceuticals (C.D.N.T., M.S.), The Frythe, Welwyn, Herts, U.K. Address correspondence and reprint requests to Dr. Robin Buckingham, Department of Vascular Biology, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park (North), Third Ave., Harlow, Essex, CM19 5AW, U.K. Received for publication 27 October 1997 and accepted in revised form 22 April 1998. ANOVA, analysis of variance; IGT, impaired glucose tolerance; NAG, Nacetyl- -D -glucosaminidase; sBP, systolic blood pressure; PPAR- , peroxisome proliferator-activated receptor- . 1326

A

ccumulating clinical evidence demonstrates that the occurrence of microalbuminuria is associated with a prediabetic insulin resistance syndrome (1–3). Because microalbuminuria is also predictive of future clinical proteinuria and increased cardiovascular mortality (4), it has been suggested that it represents an index of extrarenal macrovascular disease as well as of renal disease (5). These epidemiological data highlight the possibility that early resolution of insulin resistance in the at-risk population may not only prevent progression from the stage of impaired glucose tolerance (IGT) to type 2 diabetes, but also reverse critical cardiovascular risk factors and prevent the development of arterial and renal complications. Candidate drugs that may potentially deliver such an outcome include members of the thiazolidinedione chemical class of insulin action–enhancing agents (e.g., rosiglitazone, troglitazone, pioglitazone), which have been shown to exert antihyperglycemic and antihyperinsulinemic actions in laboratory animal models of IGT and type 2 diabetes (6–8) and in humans (9–11). The Zucker fatty (fa/fa) rat exhibits a number of the characteristics that are common to the IGT stage of the continuum leading to type 2 diabetes—that is, obesity, insulin resistance, glucose intolerance, hypertriglyceridemia, hyperinsulinemia, and hypertension. In addition, the Zucker fatty rat develops significant organ pathology, including pancreatic islet cell hyperplasia (12) and a progressive nephropathy with proteinuria (13). This is a highly exaggerated form of renal disease with no obvious clinical correlate in the prediabetic phase. Nonetheless, we cannot exclude the possibility that drugs that prevent the development and/or progression of this renal disease in the Zucker fatty rat will have beneficial effects on microalbuminuria and cardiovascular risk factors in humans. In the present study, we sought to determine the effects of long-term treatment with rosiglitazone on renal function and renal and pancreatic islet morphology of Zucker fatty rats in the belief that the outcome might have some relevance to the development and progression of damage to these organs during the clinical course of IGT. That rosiglitazone (5-{4-[(N-methyl-N-(2pyridyl) amino) ethoxy] benzyl} thiazolidine-2, 4-dione) is an insulin action-enhancing drug in Zucker fatty rats was shown in an earlier study (14). DIABETES, VOL. 47, AUGUST 1998

R.E. BUCKINGHAM AND ASSOCIATES

RESEARCH DESIGN AND METHODS Animals and drug treatment. Male Zucker fatty (fa/fa) rats and age-matched male Zucker lean (fa/?) rats (Harlan, Bicester, Oxfordshire, U.K.) were housed on a 12-h light cycle at 21 ± 2°C. At ages 6–7 weeks, the fatty rats were separated into three weight-matched groups—prevention, intervention, and control groups— comprised of 10 rats each; 15 lean control rats were also included. Each rat had its food consumption measured daily. Except for the prevention group, all rats were given powdered standard diet (R&M 1; Special Diet Services, Witham, Essex, U.K.). The prevention group was given powdered diet containing rosiglitazone maleate (SmithKline Beecham, Essex, U.K.; 50 µmol/kg of diet). Mean daily food consumption by fatty rats in the intervention and control groups was used to determine the quantity provided for prevention group rats, since rosiglitazone increases food consumption in Zucker fatty rats (15). Our aim was to prevent excessive weight gain in the prevention group from becoming a confounding factor in the experiment. After 18 weeks, when the rats were ages 24–25 weeks, the intervention group was transferred to the rosiglitazone diet. Thereafter, the fatty control group only was used to regulate the quantity of food dispensed to rats receiving the drug. The introduction of drug treatment in the intervention group was timed to coincide with conclusive evidence that control fatty rats were proteinuric. The experiment was concluded and the rats were exsanguinated during weeks 41–42, when the animals were ages 47–49 weeks. Because the protocol did not allow for adjustment of diet drug concentration with increasing body mass, there was a gradual decline in daily drug dosage per kilogram of body weight. For the prevention group, drug consumption began at 6 µmol/kg body weight at the start and declined to ~1.1 µmol/kg body weight at the end. Routine procedures. Prior to commencing the experiment, and thereafter at 3–5 week intervals, systolic blood pressure (sBP) was determined during restraint, using a noninvasive plethysmographic technique (Apollo model 179; IITC Life Science, Woodland Hills, CA) while the animals were warmed at an ambient temperature of 29–31°C. At similar intervals as were used for the sBP measurements, the rats were placed in metabolism cages (Harvard Bioscience, South Natick, MA) for 24 h and their urine was collected, weighed, and aliquotted. Aliquots of urine were frozen at –70°C for subsequent analysis of total protein (Bio-Rad protein assay; Bio-Rad, Richmond, CA), N-acetyl- -D-glucosaminidase (NAG) activity (Boehringer Mannheim test kit [Mannheim, Germany] for use on a BM/Hitachi 717 Autoanalyser (Boehringer Mannheim/Hitachi, Lewes, Sussex, U.K.) and, in some samples, albumin (Bind A Rid Rat Albumin “NL” radial immunodiffusion assay, The Binding Site, Birmingham, U.K.). Blood sampling and analyses. Blood samples were obtained from the tail tip of conscious rats, usually after a 24-h fast, at various intervals during the study. Plasma samples were stored at –70°C for later measurement of insulin (Rat Insulin [125I] assay system with Amerlex-M magnetic separation; Amersham, Little Chalfont, U.K.), triglycerides, cholesterol, and albumin (test kits supplied by Merck [Rahway, NJ], Wako [Wako Chemicals, Neuss, Germany], and Boehringer Mannheim, respectively, for use on a Molecular Devices Spectra Max 250 analyser [Molecular Devices, Crawley, Sussex, U.K.] or BM/Hitachi 717 Autoanalyser, as appropriate ). Blood glucose was determined using a 2300 Stat, Yellow Springs Glucose Analyzer (YSI, Yellow Springs, OH). Tissue analyses. At necropsy, pancreases and kidneys were removed. The pancreas from each animal was divided laterally and the proximal (head) and distal (tail) ends were subdivided longitudinally. One half of each end was weighed and placed in ice-cold acid ethanol for homogenization/extraction; the insulin content was then assayed (see above). The remaining tissue was fixed in buffered formol saline for histology (hematoxylin and eosin) or immunohistochemistry (insulin, glucagon) or was fixed in 3% glutaraldehyde for transmission electron microscopy. Kidneys were weighed and the poles were removed. Those from one kidney were frozen in liquid N2 for later histochemical/immunohistochemical analysis (alkaline phosphatase, fibronectin); the poles from the contralateral organ were placed in 0.05 N acetic acid before processing for determination of cortical cell membrane phospholipid fatty acid composition, using a modification of a previously published method (16). Briefly, the tissues were homogenized and heated at 95°C for 30 min; the homogenates were then snap frozen in liquid N2. Pellets from thawed/centrifuged (12,500 rpm) aliquots were extracted with methanol/chloroform (1:1 and 1:2) and the extracts were evaporated to near dryness under N2. Residues were derivatized with 14% boron trifluoride in methanol reagent (100°C for 10 min). Fatty acid methyl esters were extracted in hexane, evaporated to dryness under N2, and redissolved in methanol. The renal cortical fatty acid profiles of 31 animals were determined by gas chromatography/mass spectrometry. The remaining kidney tissue was fixed in buffered formol saline before histological study. Analysis of results. A split-plot analysis of variance (ANOVA), incorporating factors for animal, treatment group, time and the interaction among treatment groups and time was used to analyze the various parameters in the longitudinal DIABETES, VOL. 47, AUGUST 1998

FIG. 1. Longitudinal study of sBP in groups of Zucker rats designated as follows: lean control ( ; n = 15); fatty control ( ; n = 20 at 0–4 months, n = 10 at 5–9 months [after rosiglitazone intervention treatment commenced]); rosiglitazone prevention group ( ; n = 8–10); and rosiglitazone intervention group ( ; n = 10). Data are means ± SE. Statistical analysis notations are given on the abscissa and, like the sym bols above, are common throughout Figs. 1–3 as follows: aP < 0.05, fatty control versus lean control; bP < 0.05, fatty control versus lean control and rosiglitazone prevention groups; cP < 0.05, fatty control versus lean control, rosiglitazone prevention, and rosiglitazone intervention groups; dP < 0.05, fatty control versus rosiglitazone prevention group.

study. The residual maximum likelihood methodology (17–19) was used to calculate the ANOVA to compensate for missing values. For parameters measured only at specific times during the study (e.g., plasma insulin, triglycerides, cholesterol) or at the end (e.g., kidney weights, pancreatic insulin, renal cortical linoleic/oleic acid ratios), a one-way ANOVA was performed. Individual pairwise comparisons among selected treatment groups were performed as appropriate. These were calculated as 95% CI for the difference in means using the estimates of means and standard errors from the ANOVA. Onset of drug action in the longitudinal study was assessed by review of statistical significances across time points, along with graphic and tabular representations of the data. A 95% CI of a difference not containing zero, or for a ratio not containing 1, indicated a statistically significant difference at the 5% level (20). For studies of tissue pathology, all slides were viewed by a pathologist (C.D.N.T.) blind to the treatment groups. Morphological examination of kidneys (full transverse sections through the pelvic region of both kidneys) and pancreases was performed using a scoring system (see legend to Table 3). The scores for the four groups were compared in StatXact (Cytel Software, Cambridge, MA) using an exact Kruskal-Wallis test, and follow-up pairwise comparisons of the groups were performed in StatXact using exact Mann-Whitney U tests, with a Bonferroni approach to assessing the real significance of the pairwise tests. Following review of the findings of this scoring protocol, one animal in the fatty control group (#234) and another animal in the rosiglitazone prevention group (#220) were selected as being representative of their respective groups; the various plates in this study showing the effects of rosiglitazone treatment are taken from these rats.

RESULTS

For the sake of clarity in Figs. 1–3, data for the intervention and fatty control groups were pooled during the period (0–4 months) when both groups were receiving control diet only. Throughout this phase, there were no statistically significant differences between these groups. Statistical significances in Figs. 1–3 are derived from comparisons with the fatty control group only. Body weight. Despite having their food consumption restricted, the rosiglitazone-treated rats gained more weight than fatty control rats during the study. For example, after 39 weeks of the study, the group mean body 1327

ROSIGLITAZONE AND ZUCKER RAT

FIG. 3. Longitudinal study of urinary NAG activity in groups of Zucker rats.

FIG. 2. Longitudinal study of the urinary protein (A) and albumin (B) concentration in groups of Zucker rats.

weights (± SE) were prevention group, 797 ± 35 g (n = 8; P < 0.05 vs. fatty control); intervention group, 764 ± 15 g (n = 10; P < 0.05 vs. fatty control); fatty control group, 703 ± 24 g (n = 10); and lean control group, 468 ± 9 g (n = 15; P < 0.05 vs. fatty control). Systolic blood pressure. The sBP of fatty control rats was initially below that of lean controls (Fig. 1), but then increased rapidly and, after 4 months, was significantly elevated. In the prevention group, rosiglitazone decreased the rate of rise in sBP from 2 months onward. Commencing drug treatment at 4 months caused sBP to fall from 5 months onward. Urinary protein and albumin. Although there was evidence of proteinuria in fatty controls at the beginning of the experiment (0 months in Fig. 2A), this did not become persistent until approximately ages 18–19 weeks, at which time there was a substantial escalation in urinary protein excretion. Rosiglitazone ameliorated the proteinuria. Fig. 2A shows that, in prevention mode, rosiglitazone delayed its onset and then markedly retarded its rate of progression once established after 7 months (significant versus lean control at 7 months; separate analysis not shown on Fig. 2A). Even when 1328

given as an intervention measure, after proteinuria was established, rosiglitazone retarded progression of the disease within 1 month of the commencement of treatment. The results for proteinuria were essentially mimicked by the urinary albumin data (Fig. 2B). Rosiglitazone in the prevention group delayed the onset of albuminuria until 6 months (separate analysis for prevention versus lean control not shown in Fig. 2B) and, in both treatment modes, the drug delayed its rate of progression once established. Urinary N-acetyl- -D-glucosaminidase activity. Urinary NAG activity was raised in fatty controls compared with lean controls at 1 month and after 3 months and beyond (Fig. 3). In the rosiglitazone prevention group, NAG activity fell to around the level in lean rats from the second month onward. In the intervention group, urinary NAG activity was reduced relative to that of fatty controls from 1 month after treatment commenced (i.e., at 5 months in Fig. 3). Blood glucose, plasma insulin, lipids, and albumin. Zucker fatty control rats were hyperinsulinemic at 31 and 37 weeks and hypertriglyceridemic and hypercholesterolemic at 31 weeks (Table 1; week 37 insulin data given below) relative to lean control rats. Rosiglitazone treatment significantly reduced the plasma insulin at 31 weeks, although not at 37 weeks. At the latter time, the plasma insulin values (± SE) in each group were prevention group, 6.6 ± 1.2 ng/ml (n = 8; NS versus fatty control group); intervention group, 6.7 ± 0.8 ng/ml (n = 9; NS versus fatty control group); fatty control group, 7.8 ± 0.9 ng/ml (n = 9); and lean control group, 1.7 ± 0.2 ng/ml (n = 15; P < 0.05 versus fatty control group). Plasma total cholesterol was reduced by rosiglitazone at 31 weeks, although total triglycerides fell only in the prevention group (Table 1). Blood glucose at 37 weeks was slightly, though significantly, higher in fatty controls than in lean controls. In rosiglitazone-treated groups, the values were between those in fatty and lean controls, but were not significantly lower than in the fatty control group (Table 1). At 39 weeks, the plasma albumin concentration was markedly depleted in Zucker fatty control rats compared with in lean rats (Table 1). Consistent with the urinary albuDIABETES, VOL. 47, AUGUST 1998


TABLE 1 Blood samples taken during weeks 31 (insulin, triglycerides, cholesterol), 37 (glucose), and 39 (albumin)

Group Lean control Fatty control Rosiglitazone prevention Rosiglitazone intervention

Plasma insulin (ng/ml)

Blood glucose (mmol/l)

Plasma triglycerides (mg/dl)

Plasma cholesterol (mg/dl)

Plasma albumin (g/l)

1.2 ± 0.1* (–7.2 to –11.1) 10.4 ± 1.2 6.0 ± 0.8* (–2.3 to –6.4) 5.9 ± 0.4* (–2.4 to –6.6)

3.10 ± 0.07* (–0.25 to –0.83) 3.64 ± 0.14 3.52 ± 0.13 (–0.45 to 0.21) 3.43 ± 0.10 (–0.52 to 0.11)

82 ± 3* (–133 to –250) 274 ± 39 177 ± 15* (–35 to –159) 226 ± 17 (11 to –106)

129 ± 7* (–97 to –158) 257 ± 19 190 ± 8* (–35 to –99) 225 ± 8* (–2 to –62)

31.9 ± 0.3* (3.8–6.8) 26.6 ± 0.7 30.1 ± 0.9* (1.8–5.3) 28.8 ± 0.6* (0.6–3.8)

Data are means ± SE (95% CI) for the difference from fatty control group. A 95% CI of a difference not containing zero indicates a statistically significant difference at the 5% level (*P < 0.05).

min measurements (Fig. 2B), rosiglitazone protected against the depletion of plasma albumin, with the prevention mode being particularly effective (Table 1). Kidney weights and cortical cell membrane phospholipid fatty acid composition. Kidney mass in fatty control rats was greater than in lean control rats (Table 2), but prevention and intervention treatment with rosiglitazone blocked the development of nephromegaly. Indeed, kidney weight in the drug treatment groups was not statistically different from that of the lean control group (separate analysis not shown in Table 2). Renal cortical cell membrane phospholipid fatty acid composition was represented in terms of the linoleic acid/oleic acid ratio (Table 2), an index used previously by others (16) to define a difference between Zucker fatty and lean rats. In the lean control rats in this study, the ratio was close to unity, but in fatty control rats it was reduced by >75%. Rosiglitazone treatment in the prevention group significantly raised the linoleic/oleic acid ratio, but not to the level of lean rats. Kidney morphology, histochemistry, and immunocytochemistry. The data analysis for renal morphology is given in Table 3. Figure 4 is comprised of photographs of kidney sections exemplifying the principal renal pathological features observed in the representative fatty control rat (#234) and the prevention of these changes by rosiglitazone (rat #220). In fatty control rat kidneys, a statistically significant degree of chronic nephropathy was observed relative to lean controls (Table 3). The chronic nephropathy involved dilated tubules, often containing pink proteinaceous material (acidophilic casts) (Fig. 4A). The tubular epithelium was either flattened and atrophic or was hyperplastic, sometimes containing pro-

teinaceous (acidophilic) droplets or vacuoles. Many tubules were devoid of alkaline phosphatase activity, indicating absence of functional microvilli (not shown). Others exhibited dense alkaline phosphatase staining, consistent with a compensatory increase in the activity of brush-border microvilli of surviving structures (not shown). Tubular basement membranes were often thickened (Fig. 4A), staining densely for fibronectin (not shown), and there was a variable degree of chronic interstitial nephritis (Fig. 4A and C). The glomeruli often showed evidence of segmental (part of the tuft) or global (whole tuft) glomerulosclerosis (Fig. 4C), staining densely for fibronectin (not shown). Hydronephrosis was common in fatty control rats (5 of 10), probably due to renal parenchymal atrophy associated with advanced chronic nephropathy. In the rosiglitazone prevention group, there was a significant reduction in the incidence and degree of chronic nephropathy (Table 3), and there were no cases of hydronephrosis. Sections typifying the near-normal morphology of renal cortex and glomerulus in this group are shown in Fig. 4B and D, respectively. Also, in contrast with the fatty control group, the tubular basement membranes and glomeruli stained only lightly for fibronectin in the rosiglitazone prevention representative rat (not shown). The presence of abundant alkaline phosphatase activity in this animal confirmed the normality of the microvillous border and the tubular epithelial cells (not shown). In the rosiglitazone intervention group, there was a reduction in the degree of chronic nephropathy compared with the fatty control group, but this was greater than that seen in the prevention group and was not statistically significant (Table 3). Mild hydronephrosis was observed in 2 of 10 rats.

TABLE 2 Data on kidneys and pancreases were removed from Zucker rats during weeks 41–42 of the experiment Group Lean control Fatty control Rosiglitazone prevention Rosiglitazone intervention

Combined weight of kidneys (g) 2.78 ± 0.05* (–1.19 to –0.78) 3.77 ± 0.11 2.69 ± 0.09* (–1.30 to –0.85) 2.95 ± 0.07* (–1.04 to –0.60)

Renal cortical cell linoleic/oleic acid ratio

Pancreatic insulin content (ng/mg tissue)

0.93 ± 0.03* (0.60 to –0.82) 0.22 ± 0.03 0.36 ± 0.02* (0.04 to –0.24) 0.32 ± 0.04 (–0.002 to 0.19)

138 ± 8* (–61 to –174) 255 ± 30 289 ± 25 (–32 to 99) 290 ± 25 (–27 to 97)

Data are means ± SE (95% CI) for the difference from fatty control group. A 95% CI of a difference not containing zero indicates a statistically significant difference at the 5% level (*P < 0.05). DIABETES, VOL. 47, AUGUST 1998

1329


TABLE 3 Data analysis for renal and pancreatic morphology

Group Fatty control Lean control

CN 0.0001 †

Lean control HN HP 0.232 NS

0.0001 †

IN 0.0001 †

Rosiglitazone prevention CN HN HP IN 0.0002 † 0.9974 NS

0.0359 NS 0.0582 NS

0.0001 † 1.0000 NS

Rosiglitazone prevention

0.0001 † NT

Rosiglitazone intervention CN HN HP IN 0.0085 S 0.0100 S 0.0106 S

0.0712 NS 0.3497 NS 0.4771 NS

0.0019 0.0001 * † 0.0008 NT † 0.0282 NT NS

Tissue samples were scored blind by a pathologist (C.D.N.T.). In the kidney, scores were generated for chronic progressive nephropathy (CN) and hydronephrosis (HN). In pancreatic islets, scores were generated for the presence of cellular hyperplasia (HP) and insulin content (IN). In each case, the features were scored by degree as follows: 0, none seen; 1, minimal; 2, mild; 3, moderate; 4, marked. Data were then analyzed by the Kruskal-Wallis test and exact Mann-Whitney U test for multiple comparisons. P values in the table are equal to or less than those given. Conclusions regarding significance are made using the Bonferroni approach as follows: NT, no statistical comparison made because the scores were identical across the groups; NS, not significant; S, some evidence of difference, but not statistically significant; *, significantly different; †, strongly significantly different.

The normal background renal pathology for this experiment was provided by the age-matched lean controls. Even in this group, a degree of chronic nephropathy was seen in 11 of 15 rats and hydronephrosis was seen in 6 rats. Overall, these results demonstrated that the lean control and the rosiglitazone prevention groups scored similarly for the incidence and degree of nephropathy, although the mild hydronephrosis observed in some lean controls was not seen in the prevention group. Pancreatic insulin content, histology, immunocytochemistry, and transmission electron microscopy. Pancreatic tissue insulin content was higher in fatty control rats than in lean control rats, but treatment of fatty rats with rosiglitazone did not influence insulin concentration (Table 2). The data analysis for pancreatic morphology is shown in Table 3. Figure 5 is comprised of photographs exemplifying the principal pancreatic pathological and immunocytochemical features in the representative fatty control rat (#234) and the effect of drug treatment with rosiglitazone in the prevention mode (rat #220). Islet cell hyperplasia, fine vacuolation, and hypertrophy of varying severity were noted in all fatty control animals. Characteristically, the enlarged islets had a disorganized morphology, with expansion into adjacent exocrine tissue (Fig. 5A). The -cells had a reduced insulin content (Table 3), as demonstrated by the light immunocytochemical staining in Fig. 5C, and glucagon-containing -cells were seen as single cells or small groups of cells scattered throughout the hyperplastic islets (Fig. 5E). This disseminated distribution of cells suggests trapping as islands within a matrix of dividing islet cells. Ultrastructurally, -cells from fatty control rats had a relative paucity of secretory granules, hypertrophy of the Golgi apparatus, hyperplasia of the rough endoplasmic reticulum, and dilation of the endoplasmic reticulum (not shown). There was a good correlation between these ultrastructural changes, light microscopy, and the immunocytochemical staining for insulin. There were no abnormal ultrastructural changes within the -cells of the fatty control rat (not shown). In the rosiglitazone prevention group, islet morphology was essentially normal (Table 3, Fig. 5B), except for a few islets with a minimal degree of hyperplasia. 1330

The insulin content of -cells was high (Table 3), as demonstrated by dense immunocytochemical staining in Fig. 5D, and the -cells were located peripherally (Fig. 5F). -Cell ultrastructure was normal, with an abundance of insulin secretory granules present in the cytoplasm (not shown). In the rosiglitazone intervention group, there was an intermediate islet pathology with a mild islet hyperplasia in 5 of 10 rats; a further 4 animals exhibited no or minimal changes (Table 3). -Cells were lightly staining, not hypertrophied or vacuolated, and had a high insulin content. The majority of cells were located around the islet periphery. In contrast to the fatty control group, islets from lean controls showed a normal morphology (Table 3). Only 2 of 10 lean control rats showed any sign of islet cell hyperplasia, and these were scored as minimal. DISCUSSION

These data chronicle the onset and progression of renal disease in Zucker fatty rats and demonstrate that rosiglitazone markedly reduces renal injury in this model. The earliest marker of impending nephropathy in fatty control rats was a rise in their urinary NAG activity, an enzyme found in lysosomes of renal proximal tubular cells. Evidence of a marginal albuminuria at 2 months suggested that the onset of functional glomerular damage occurred at ages 13–19 weeks, according well with published data (13,21,22). Onset of proteinuria in fatty controls coincided with the development of hypertension, suggesting that high blood pressure itself did not initiate early glomerular damage, a finding also consistent with the literature (23–25). Rosiglitazone is a potent insulin action–enhancing agent of the thiazolidinedione chemical class (6,14,26,27), presently undergoing clinical testing for the treatment of type 2 diabetes (9). In the rosiglitazone prevention group in this study, there was early normalization of urinary NAG activity, a prolonged delay in the onset of proteinuria and albuminuria, and prevention of hypertension. Even when proteinuria/albuminuria did become established in this group, it progressed more gradually than in fatty controls. When given in the intervention mode, after the establishment of proteinuria, rosiglitazone again normalized urinary NAG activity, prevented further elevation of blood pressure, and DIABETES, VOL. 47, AUGUST 1998


FIG. 4. Kidney cortex: hematoxylin and eosin staining. Original magnification 200: A: Zucker fatty control rat #234 exhibits moderate degree of chronic nephropathy; B: rosiglitazone prevention treatment of fatty rat #220: normal morphology shown. Original magnification 800: C: Zucker fatty control rat #234 showing a single glomerulus with a moderate degree of global glomerular sclerosis; D: rosiglitazone prevention treatment of fatty rat #220 demonstrating normal “open-lattice” morphology of a single glomerulus.

reduced the rate of escalation of proteinuria, suggesting that although preexisting glomerular injury could not be reversed, its progression could be retarded. The ability of rosiglitazone to protect against glomerular damage was also reflected in its preventive effect against the depletion of plasma albumin. From the earlier observation on the coincidental onset of proteinuria and hypertension in fatty control rats, it is possible that the antihypertensive action of rosiglitazone is at least partly due to its ability to prevent or arrest structural renal damage. Our observations on renal pathology in fatty control rats confirm published observations with respect to nephromegaly, glomerulosclerosis (13), increased glomerular fibronectin deposition (28), tubular dilation and interstitial fibrosis (21,29), and a reduced linoleic acid/oleic acid ratio in cortical cell membrane phospholipids (16). In addition, our observations supplement the literature concerning tubular injury (21,29), demonstrating increased fibronectin staining in basement membranes and a loss of functional epithelial microvilli. Our results also show a high prevalence of hydronephrosis in aged Zucker fatty rats, presumably due to escalating tubular atrophy, attributable to blockage of nephrons by proteinaceous casts. DIABETES, VOL. 47, AUGUST 1998

This picture of severe renal disease in fatty controls contrasts with that in rosiglitazone-treated rats. In the prevention group, kidney mass and pathology were indistinguishable from that of lean controls. Nonetheless, there was a mild progressive proteinuria, suggesting that rosiglitazone did not completely prevent functional glomerular damage, possibly due to a gradual decline in the daily rosiglitazone dosage. Data from other experiments suggest that rosiglitazone, 3 µmol/kg body weight daily, normalizes glucose tolerance in Zucker fatty rats (14), but that a dosage of 0.3 µmol/kg is ineffective (S.A. Smith, unpublished observations). In the present study, there was no longer a significant antihyperinsulinemic effect of rosiglitazone at 37 weeks, when the daily dosage was substantially below 2 µmol/kg body weight, a finding that tends to support this hypothesis. In the intervention group, evidence of chronic nephropathy was more prominent but still less severe than in fatty control rats. This intermediate pathology was consistent with renal damage inflicted before drug treatment being irreversible. Although the present data have not identified the mechanism of rosiglitazone’s renal protective action, there are a number of possible candidates: 1) Rosiglitazone partially corrected the diminished availability of polyunsaturated fatty acids in cortical cell membranes, an effect that may have a 1331


FIG. 5. Pancreas. Original magnification 125, hematoxylin and eosin staining: A: Zucker fatty control rat #234 showing a moderate degree of islet hyperplasia and fibrosis; B: rosiglitazone prevention treatment of fatty rat #220: normal islet morphology. Insulin staining (indirect immunoperoxidase technique): C: Zucker fatty control rat #234 showing a variable density of insulin staining and uneven distribution of cells; D: rosiglitazone prevention treatment of fatty rat #220: high density of insulin staining and a normal distribution of -cells. Original magnification 200; glucagon staining (indirect immunoperoxidase technique): E: Zucker fatty control rat #234 showing -cells distributed throughout the hyperplastic islet; F: rosiglitazone prevention treatment of fatty rat #220: normal distribution of -cells at the periphery of the islet.

bearing on membrane fluidity and function (16). 2) Rosiglitazone markedly reduced fibronectin deposition. Although the mechanism of this action is unknown, proteolytic activity against intraglomerular fibronectin is reduced in Zucker fatty rats (28), a deficiency that potentially leads to increased adherence and motility of fibroblasts (30). 3) Rosiglitazone had an antihyperlipidemic effect that, although modest, may have had a bearing on the renal pathology (22,24,25,31–34). 1332

4) The demonstration (35) that the drug is a high-affinity and selective ligand for peroxisome proliferator-activated receptor- (PPAR- ), a member of the nuclear receptor superfamily that promotes pre-adipocyte differentiation and adipogen esis and which has the effect of lowering plasma free fatty acids, has raised the possibility that this molecular target may constitute a common mediator for rosiglitazone’s actions. Although PPAR- is expressed in low abundance in DIABETES, VOL. 47, AUGUST 1998


the renal cortex (glomerulus, proximal tubule) (36), nothing is known about its functional role in this tissue; the possibility that rosiglitazone exerts a renal protective action via a direct effect on this receptor in the kidney cannot be excluded. Because rosiglitazone is an insulin action–enhancing agent in Zucker fatty rats (14), and here we have shown it to reduce the fasting plasma insulin concentration at 31 weeks (though not at 37 weeks), we also studied the pancreatic islet pathology. As with the kidneys, there were pancreatic morphological abnormalities of Zucker fatty control rats, changes that were ameliorated or prevented by rosiglitazone. Islet -cell hyperplasia (12), an elevated pancreatic insulin content (37,38), and a disseminated distribution of -cells throughout the islet (12) have each been described previously in fatty rats and were prominent features here. The signature of work hypertrophy (i.e., Golgi hypertrophy, hyperplasia of rough endoplasmic reticulum, and dilation of endoplasmic reticulum) was also apparent at the ultrastructural level of the cell. The beneficial effects of rosiglitazone on islet pathology are presumably a direct consequence of the drug’s antihyperinsulinemic action, interrupting the cycle of escalating tissue insulin resistance and the compensatory increase in insulin secretion. However, there remains a conundrum: whereas islets in rosiglitazone prevention group rats were morphologically similar to those in lean rats, and their cells appeared normal ultrastructurally, the pancreatic tissue insulin concentration was similar to that in fatty control rats. This suggests that stored insulin was unrelated to differences in insulin secretory demand in the two groups. Previous experiments in Zucker fatty rats in our laboratory have shown that rosiglitazone reduced islet pre-proinsulin mRNA but did not influence systemic clearance of the hormone (S.A. Smith, unpublished observations). Those data imply that the reduced secretory demand for insulin is matched by reduced synthesis. Although these parameters were not measured in the present study, we speculated that total insulin storage might be a constant in adult Zucker fatty rats, irrespective of islet morphology. In conclusion, our results support the rationale for testing insulin action–enhancing agents in IGT in humans to determine whether they might reduce or prevent progression to type 2 diabetes. The renal protective effect of rosiglitazone in the Zucker fatty rat, a severe model of the prediabetic insulin resistance syndrome, may also imply that insulin action–enhancing agents will prove to be effective in inhibiting the onset or reducing the rate of progression of renal disease in insulin-resistant, prediabetic individuals and in patients with type 2 diabetes, in whom elements of this metabolic syndrome are prevalent. REFERENCES 1. Niskanen L, Laakso M: Insulin resistance is related to albuminuria in patients with type II (non-insulin-dependent) diabetes mellitus. Metabolism 42:1541–1545, 1993 2. Haffner SM, Stern MP, Gruber MKK, Hazuda HP, Mitchell BD, Patterson JK: Microalbuminuria: potential marker for increased cardiovascular risk factors in nondiabetic subjects? Arteriosclerosis 10:727–731, 1990 3. Haffner SM, Gonzales C, Valdez RA, Mykkanen L, Hazuda HP, Mitchell BD, Monterrosa A, Stern MP: Is microalbuminuria part of the prediabetic state? The Mexico City Diabetes Study. Diabetologia 36:1002–1006, 1993 4. Mogensen CE: Microalbuminuria predicts clinical proteinuria and early mortality in maturity-onset diabetes. N Engl J Med 310:356–360, 1984 5. Yudkin JS, Forrest RD, Jackson CA: Microalbuminuria as predictor of vascular disease in non-diabetic subjects. Lancet 1:530–533, 1988 6. Young PW, Cawthorne MA, Coyle PJ, Holder JC, Holman GD, Kozka IJ, DIABETES, VOL. 47, AUGUST 1998

Kirkham DM, Lister CA, Smith SA: Repeat treatment of obese mice with BRL 49653, a new and potent insulin sensitizer, enhances insulin action in white adipocytes: association with increased insulin binding and cell surface GLUT 4 as measured by photoaffinity labeling. Diabetes 44:1087–1092, 1995 7. Sreenan S, Sturis J, Pugh W, Burant CF, Polonsky KS: Prevention of hyperglycemia in the Zucker diabetic fatty rat by treatment with metformin or troglitazone. Am J Physiol 271:E742–E747, 1996 8. Kemnitz JW, Elson DF, Roecker EB, Baum ST, Bergman RN, Meglasson MD: Pioglitazone increases insulin sensitivity, reduces blood glucose, insulin, and lipid levels, and lowers blood pressure in obese, insulin-resistant rhesus monkeys. Diabetes 43:204–211, 1994 9. Patel J, Miller E, Hu J, Granett J: BRL 49653 (a thiazolidinedione) improves glycemic control in NIDDM patients (Abstract). Diabetes 46 (Suppl. 1):150A, 1997 10. Iwamoto Y, Kuzuya T, Matsuda H, Awata T, Kumakura S, Inooka G, Shiraishi I: Effect of new oral antidiabetic agent CS-045 on glucose tolerance and insulin secretion in patients with NIDDM. Diabetes Care 14:1083–1086, 1991 11. Wasada T, Omori Y, Sasaki H, Kawamori R, Yamasaki Y, Baba S, Shichiri M, Kaneko T: Effect of pioglitazone (AD-4833) on insulin-stimulated glucose disposal in NIDDM: an assessment by the euglycemic hyperinsulinemic clamp method. Diabetes 45 (Suppl. 2):73A, 1996 12. Larsson L-I, Boder GB, Shaw WN: Changes in the islets of Langerhans in the obese Zucker rat. Lab Invest 36:593–598, 1977 13. Kasiske BL, Cleary MP, O’Donnell MP, Keane WF: Effects of genetic obesity on renal structure and function in the Zucker rat. J Lab Clin Med 106:598–604, 1985 14. Smith SA, Coyle PJ, Cawthorne MA, Holder JC, Kirkham D, Lister CA, Murphy GJ, Young PW: BRL 49653 normalises glycaemic control in Zucker fa/fa rats by improving hepatic and peripheral sensitivity to insulin. Diabetologia 36 (Suppl. 1):A184, 1993 15. Wang Q, Dryden S, Frankish HM, Bing C, Pickavance L, Hopkins D, Buckingham R, Williams G: Increased feeding in fatty Zucker rats by the thiazolidinedione BRL 49653 (rosiglitazone) and the possible involvement of leptin and hypothalamic neuropeptide Y. Br J Pharmacol 122:1405–1410, 1997 16. Kasiske BL, O’Donnell MP, Cleary MP, Keane WF: Effects of reduced renal mass on tissue lipids and renal injury in hyperlipidemic rats. Kidney Int 35:40–47, 1989 17. Patterson HD, Thompson R: Recovery of inter-block information when block sizes are unequal. Biometrika 58:545–554, 1971 18. Robinson DL, Thompson R, Digby PGN: REML: a program for the analysis of non-orthogonal data by restricted maximum likelihood. Compstat 1982. Pro ceedings in Computational Statistics, Part II (Suppl). Vienna, Physica Verlag, 1982, p. 231–232 19. Genstat 5 Committee of the Statistics Department, Rothamsted Experimental Station: Genstat 5 Release 3 Reference Manual. Payne RW, Lane PW, Eds. Oxford Science Publications, Oxford, U.K., Clarendon Press, 1993, p. 539–583 20. Senn SJ, Auclair P: The graphical representation of clinical trials with particular reference to measurement over time. Stat Med 9:1287–1302, 1990 21. Magil AB: Tubulointerstitial lesions in young Zucker rats. Am J Kidney Dis 25:478–485, 1995 22. Kamanna VS, Kirschenbaum MA: Association between very-low-density lipoprotein and glomerular injury in obese Zucker rats. Am J Nephrol 13:53–58, 1993 23. O’Donnell MP, Kasiske BL, Cleary MP, Keane WF: Effects of genetic obesity on renal structure and function in the Zucker rat. II. Micropuncture studies. J Lab Clin Med 106:605–610, 1985 24. Schmitz PG, O’Donnell MP, Kasiske BL, Katz SA, Keane WF: Renal injury in obese Zucker rats: glomerular haemodynamic alterations and effects of enalapril. Am J Physiol 263:F496–F502, 1992 25. Crary GS, Swan SK, O’Donnell MP, Kasiske BL, Katz SA, Keane WF: The angiotensin II receptor antagonist losartan reduces blood pressure but not renal injury in obese Zucker rats. J Am Soc Nephrol 6:1295–1299, 1995 26. Cantello BCC, Cawthorne MA, Haigh D, Hindley RM, Smith SA, Thurlby PL: The synthesis of BRL 49653—a novel and potent antihyperglycaemic agent. Bioorg Med Chem Lett 4:1181–1184, 1994 27. Cantello BCC, Cawthorne MA, Cottam GP, Duff PT, Haigh D, Hindley RM, Lister CA, Smith SA, Thurlby PL: [[ -(Heterocyclylamino)alkoxy]benzyl]-2,4-thiazolidinediones as potent antihyperglycemic agents. J Med Chem 37:3977–3985, 1994 28. Paczek L, Teschner M, Schaefer RM, Heidland A: Intraglomerular fibronectin accumulation and degradation in obese Zucker rats. Diabetologia 34:786–789, 1991 29. Magil AB, Frohlich JJ: Monocytes and macrophages in focal glomerulosclerosis in Zucker rats. Nephron 59:131–138, 1991 30. Gauss-Muller V, Kleinmann HK, Martin GR, Schiffmann E: Role of attachment fac1333


tors and attractants in fibroblast chemotaxis. J Lab Clin Med 96:1071–1080, 1980 31. Kasiske BL, O’Donnell MP, Cleary MP, Keane WF: Treatment of hyperlipidemia reduces glomerular injury in obese Zucker rats. Kidney Int 33:667–672, 1988 32. O’Donnell MP, Kasiske BL, Kim Y, Schmitz PG, Keane WF: Lovastatin retards the progression of established glomerular disease in obese Zucker rats. Am J Kidney Dis 22:83–89, 1993 33. Yoshioka S, Nishino H, Shiraki T, Ikeda K, Koike H, Okuno A, Wada M, Fujiwara T, Horikoshi H: Antihypertensive effects of CS-045 treatment in obese Zucker rats. Metabolism 42:75–80, 1993 34. Kasiske BL, O’Donnell MP, Lee H, Kim Y, Keane WF: Impact of dietary fatty acid supplementation on renal injury in obese Zucker rats. Kidney Int 39:1125–1134, 1991

1334

35. Lehman JM, Moore LB, Smith-Oliver TA, Wilkison WO, Willson TM, Kliewer SA: An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor (PPAR ). J Biol Chem 270:12953–12956, 1995 36. Braissant O, Foufelle F, Scotto C, Dauca M, Wahli W: Differential expression of peroxisome proliferator-activated receptors (PPARs): tissue distribution of PPAR- , - , and - in the adult rat. Endocrinology 137:354–366, 1996 37. Lemmonier D, Aubert R, Suquet J-P, Rosselin G: Metabolism of genetically obese rats on normal or high-fat diet. Diabetologia 10:697–701, 1974 38. Laburthe M, Rancon F, Freychet P, Rosselin G: Glucagon and insulin from lean rats and genetically obese fatty rats: studies by radioimmunoassay, radioreceptorassay and bioassay. Diabetologia 11:517–526, 1975

DIABETES, VOL. 47, AUGUST 1998