The role of hyperglycemia in the pathogenesis of. Kidney Morphology in Experimental Hyperglycemia

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1 Kidney Morphology in Experimental Hyperglycemia TIMOTHY S. KERN AND RONALD L. ENGERMAN SUMMARY To evaluate the role of hyperglycemia in the pathogenesis of diabetic nephropathy, the kidneys from dogs experimentally galactosemic for 5 yr have been compared with the kidneys from age-matched normal dogs and dogs with alloxan-induced diabetes for 5 yr. The width of glomerular capillary basement membrane and the quantity of plasma protein immunohistochemically demonstrable in the basement membrane were supranormal in the galactosemics, as they were in the diabetics. In contrast, kidney weight, mesangial volume, and the prevalence of obliterated glomeruli, glomerular exudates, and mesangial nodules in the galactosemic animals were comparable to those of normal animals and clearly were less than observed in the insulin-deficient diabetic animals. These galactosemic dogs are known to have developed a retinopathy morphologically indistinguishable from that of diabetic patients and dogs. Thus, galactosemia sufficient to produce diabetic-like lesions in the glomerular basement membrane and retina was found to be nevertheless insufficient to elicit several renal abnormalities that are typical of diabetes. The polyol concentration in erythrocytes was greater than normal in the galactosemics and the diabetics and was greatest in the galactosemics. The absence of mesangial expansion, glomerular obliteration, and nephromegaly in galactose-fed dogs raises the possibility that these abnormalities in diabetes are not a result of excessive polyol pathway activity. Diabetes 36:244-49, 1987 The role of hyperglycemia in the pathogenesis of diabetic glomerulopathy is not clear. Good glycemic control of diabetes reportedly can inhibit the development of glomerular lesions in experimentally diabetic animals (1-5), but hyperglycemia is only one of many metabolic abnormalities that tend to be normalized with improved control. The possible role of hyperglycemia in the etiology of glomerulopathy has remained speculative, in part because the histopathologic consequences of hyperglycemia have proven difficult to differentiate from those of the other metabolic abnormalities of insulin deficiency. Experimental galactosemia induces diabetic-like lesions in lens, cornea, peripheral nerve, and retina. Experimentally galactosemic dogs develop a retinopathy that is morphologically indistinguishable from that seen in diabetic patients and diabetic dogs (6). This retinopathy develops in the apparent absence of many metabolic disorders typical of diabetes, e.g., subnormal blood concentrations of insulin and supranormal concentrations of glucose, lipids, and branched-chain amino acids (7). The evidence strongly suggests that excessive blood hexose concentration plays a key role in the development of diabetic microvascular disease, at least in the retina. Diabetic dogs develop morphologic lesions characteristic of diabetic nephropathy in patients, including nephromegaly and, in the glomeruli, mesangial expansion, nodular mesangial deposits, exudative deposits, glomerular obliteration, and thickening of basement membrane (8-10). To evaluate the role of hyperglycemia in the development of diabetic nephropathy, the influence of experimental galactosemia on renal morphology has been evaluated in dogs and compared with the effects of diabetes. MATERIALS AND METHODS Healthy adult dogs were assigned randomly to be made experimentally galactosemic or diabetic or to remain normal control animals. Four animals have been killed after 5 yr of experimental galactosemia. Age-matched normal dogs (N = 7) and alloxan-diabetic dogs (A/ = 6) have been studied concurrently with the galactosemic animals and likewise killed for comparison after 60 mo of study. Diabetes was induced with alloxan (4), and the diabetic animals received From the Department of Ophthalmology, University of Wisconsin, Madison, Wisconsin. Address correspondence and reprint requests to Dr. T.S. Kern, Department of Ophthalmology, University of Wisconsin, 1300 University Avenue, Madison, Wl Received for publication 26 September 1986 and accepted in revised form 17 October DIABETES, VOL. 36, FEBRUARY 1987

2 S. KERN AND R. L. ENGERMAN B daily injections of insulin insufficient to prevent chronic hyperglycemia and glucosuria. The experimental galactosemia was produced by feeding the dogs a commercial diet supplemented with 30% D-galactose (6). Diabetic and normal animals received the diet without galactose supplement. Protein content of the unsupplemented diet was 26%. All animals were housed in metabolism cages, and 24-h urine samples were tested for volume, glucose, and reducing sugar several times weekly. HbA! was measured periodically with minicolumns (Isolab, Akron, OH) after incubation (4 h at 37 C) to remove labile adducts. Erythrocyte polyol concentration was measured enzymatically (sorbitol dehydrogenase) in normal and diabetic animals. Erythrocyte galactitol, undetected by that method, was quantified by gas chromatography (11). The two methods yield similar results for erythrocyte sorbitol concentration. Blood glucose and plasma galactose were measured enzymatically (6). The kidneys were obtained surgically from animals anesthetized with pentobarbital (25 mg/kg i.v.) and killed a few minutes DIABETES, VOL. 36, FEBRUARY 1987 FIG. 1. Representative glomeruli of normal dogs (A) or dogs having experimental galactosemia (B) or alloxan-diabetes (C) of 5-yr duration. Glomerulosclerosis (including mesangial expansion and glomerular exudates) and glomerular enlargement are apparent only in the glomerulus from diabetic dog. PAS and hematoxylin; (x235). later. Tissues were fixed in 10% buffered formalin or 4% glutaraldehyde or were frozen on dry ice. Histologic studies of kidney. All histologic and immunohistochemical evaluations were performed with coded specimens to minimize bias. Paraffin sections (4 xm) of kidney were stained with periodic acid-schiff (PAS) and hematoxylin and hematoxylin plus eosin. Glomerular morphology was evaluated in 25 glomeruli/animal; to ensure that glomeruli were studied near their center, only glomeruli with tuft diameters >100 (xim were evaluated. The percentage of glomerular tuft occupied by mesangium (fractional area of mesangium) was estimated semiquantitatively in tissue sections by comparing mesangial area of each glomerulus to a series of photographs illustrating increasing amounts of mesangial area, from 1 + (little mesangial area) to 6 + (much mesangium, obliterated glomerulus). Average diameter of glomerular capsule and tuft were determined from 25 consecutive glomeruli regardless of size. The frequency of obliterated (sclerotic) glomeruli, or glomeruli containing syn- 245

3 KIDNEY MORPHOLOGY IN EXPERIMENTAL HYPERGLYCEMIA TABLE 1 Glomerular histology and kidney weight in normal, experimentally galactosemic, and diabetic dogs (means ± SD) Mesangial area* Obliterated glomeruli (%) Tuft diameter ( xm) Kidney weight '(g) (g/kg) Capillary basement membrane width (nm) Normal (N = 7) 2.0 ± ± ± ± ± ± 38 (N = 5) *Semiquantitative units; see text. fp <.05 compared with normal. $P <.05 compared with galactosemia. Galactosemia (A/= 4) 2.2 ± ± ± ± ± ± 104f (N = 4) Diabetes (N = 6) 3.5 ± 0.6ft 11.4 ± 9.9ft 168 ± 16ff 44 ± 10ft 4.1 ± 0.6ft 618 ± 65f (N = 4) echia, PAS-positive exudates, or mesangial nodules, was determined from all glomeruli available in the tissue section (mean = 232 glomeruli/animal). Basement membrane thickness of peripheral glomerular capillary loops was measured by an orthogonal intercept method in representative animals from each experimental group (12). Approximately 200 measurements from each of three glomeruli were obtained from evenly spaced electron photomicrographs over the entire glomerular tuft (final magnification x 17,000-18,000). Vacuoles in glomerular basement membrane were not excluded from the measurement. Cryosections of kidney cortex from two galactosemic, two diabetic, and two normal dogs were cut (4 (JUTI) and immunostained together to localize albumin, IgG, or C 3 (complement). The sections were fixed in acetone, washed, reacted briefly with 1% normal rabbit sera, incubated with antibody against dog albumin (Cappel, Malvern, PA; 1:13,500 dilution), IgG (Miles, Naperville, IL; 1:4500 dilution), or C 3 (Miles; 1:500 dilution) for 1 h, washed again, and incubated for an additional hour in fluorescein-labeled goat anti-rabbit IgG (Sigma, 1:60 dilution). Controls included substitution of nonimmune rabbit sera for the primary antibody and omission of primary antibody. Sections were treated to prevent fading of fluorescence (13), and the intensity of fluorescence was scored semiquantitatively (1 + to 4 + ). Group comparisons were made with the Kruskal-Wallis H test and Mann-Whitney U test due to the limited sample size and semiquantitative nature of some measurements. RESULTS Kidneys of the dogs experimentally galactosemic for 5 yr seemed relatively normal on the basis of light microscopy and kidney weight (Fig. 1 and Table 1). The fraction of glomerulus occupied by mesangium was not significantly different from normal in the galactose-fed dogs. Glomerular tuft diameter, likewise, seemed normal in the galactosemics, suggesting that the total mesangial volume (calculated from fractional mesangial area and tuft diameter) was not abnormal. Nodular mesangial deposits were never observed in the glomeruli of the galactosemic or normal dogs, and little or no arteriosclerosis was detected in any group. The prevalence of obliterated glomeruli in the galactosemics was comparable to normal. Kidney weight, expressed as absolute weight or relative to body weight, was not significantly different from normal in the galactose-fed dogs. Leukocytes were observed in the cortical interstitium of only one of the four galactosemic animals. In contrast with the absence of mesangial abnormalities and nephromegaly in the galactosemic animals, the thickness of glomerular capillary basement membrane was significantly greater than normal in the same animals. Study of renal tissue from dogs diabetic for 5 yr confirmed the expected occurrence of glomerulopathy in that animal model. Abnormalities observed in glomeruli of the diabetic dogs included a supranormal tuft diameter and fractional area of mesangium, thus indicating greater than normal mesangial volume. In addition, thediabetics had excessive numbers of obliterated glomeruli and PAS-positive exudates in the tuft and/or capsule and numerous leukocytes in the interstitium. Nodular deposits of mesangium were observed in diabetic glomeruli but were infrequent. Glomerular capillary basement membrane in the diabetics was significantly thicker than that in normals and tended to be thicker also than that in the galactosemics. Nephromegaly was evident in diabetes regardless of whether kidney size was expressed as absolute weight or weight relative to body weight. Albumin, IgG, and C 3 were identified immunohistochemically in glomeruli of all animals studied. Albumin and, to a lesser extent, IgG were localized in a linear pattern along glomerular and tubular basement membranes. Slight immunostain occurred, possibly in the mesangium. C 3 was found to have a granular diffuse distribution in glomeruli. The intensity of albumin, IgG, and C 3 immunostain was slight in glomeruli of normal animals and was greater than normal in glomeruli of both the galactosemics and diabetics (Fig. 2). Galactosemic animals had slightly less glomerular albumin and C 3 than the diabetics. It was not clear whether galactosemic dogs had less IgG than the diabetic dogs. More of the glomerulus tended to be immunostained in the diabetics than in the normals or galactosemics. The galactose-fed dogs showed supranormal blood galactose concentrations (of <250 mg/dl) and daily glycosuria as expected. The galactosemic dogs and diabetic dogs had significantly greater than normal concentrations of HbA, in blood and of reducing sugar in urine as well as supranormal urine volume (P <.05; Table 2). The erythrocyte polyol concentration in galactose-fed dogs was many times greater than that in the diabetic or normal dogs, consistent with a larger study reported previously (14). All galactosemics and diabetics developed cataracts. Food consumption in the galactosemic animals was 30% greater than normal throughout the 5 yr of study (22.7 ± 3.5 vs ± 1.4 g kg" 1 day^1, P<.05), an increase that seems to have compensated for the galactose added to the diet. Food consumption in diabetic animals (42.9 ± 4.9) exceeded that of either the normal (P <.001) or galactosemic dogs (P <.01). DISCUSSION Experimental galactosemia induces various diabetic-like lesions, including retinopathy. Retinopathy in the galactosemic dogs reported herein includes saccular microaneurysms, obliterated and acellular capillaries, pericyte "ghosts," hemorrhage, and basement membrane thickening (7). The evidence suggests that excessive blood hexose concentration 246 DIABETES, VOL, 36, FEBRUARY 1987

4 S KERN AND R. L. ENGERMAN FIG. 2. Immunofluorescence micrographs of glomeruli from normal dog (A) and from experimentally galactosemic (8) and alloxan-diabetic (C) dogs stained with rabbit anti-dog albumin followed by fluorescein-labeled goat anti-rabbit IgG. Glomeruli from both galactosemic and diabetic dogs have supranormai amounts of immunostain; (x368). DIABETES, VOL. 36, FEBRUARY

5 KIDNEY MORPHOLOGY IN EXPERIMENTAL HYPERGLYCEMIA TABLE 2 Urine and blood chemistry in normal, experimentally galactosemic, and diabetic dogs (means ± SD) Glycosuria (g/dl) Urine volume (ml kg" 1-24 rr 1 ) Hemoglobin A, (%) Red blood cell polyol (nmol/g Hb) *N = 2. Normal (N = 7) 0 ± 0 15 ± ± ± 14* Galactosemia (N = 4) 4.4 ± ± ± ± 55* Diabetes (N = 6) 6.4 ± ± ± ± 17* is an important determinant of diabetic retinopathy, but its role in the development of diabetic nephropathy is less clear. Galactosemic dogs were found by us to be free of some typical features of diabetic nephropathy, i.e., glomerular obliteration, excessive mesangial volume, and renal hypertrophy. Mesangial expansion and a subsequent reduction in glomerular capillary filtering surface are believed to play an important role in the development of renal dysfunction in diabetes (15). The findings in galactosemic dogs suggest that the mesangial expansion, glomerular obliteration, and renal hypertrophy observed in diabetes might be due to abnormalities other than excessive blood hexose concentration. The polyol pathway utilizes both glucose and galactose and has been implicated in the etiology of several complications of diabetes and experimental galactosemia, including cataract, defects in nerve conduction velocity and axon transport, and thickening of capillary basement membrane (16). Excessive production or accumulation of polyol by glomeruli likewise has been postulated to play a role in the development of diabetic nephropathy. Aldose reductase, a major enzyme in the reduction of hexoses to polyols, has been detected immunohistochemically in the glomerular epithelium of rats (17), and a pharmacologic inhibitor of the enzyme has been reported to inhibit the development of microproteinuria and loss of glomerular myo-inositol and Na + -K + -ATPase activity in rats with streptozocin-induced diabetes up to 3 mo (18,19). Dog glomeruli have not been found to contain immunoreactive aldose reductase (20). Polyol production in experimentally galactosemic dogs is as great or greater than that in diabetic dogs; polyol concentration in erythrocytes of galactosemic dogs exceeds that in diabetic dogs by severalfold (14). The elevation of polyol concentration in erythrocytes reportedly is proportional to that in other tissues such as lens and nerve (21). The evidence presented herein raises the possibility that excessive polyol-pathway activity is not a major factor in the development of mesangial expansion, glomerular obliteration, and nephromegaly in diabetes inasmuch as these anatomic abnormalities were absent from galactosemic dogs having erythrocyte polyol concentrations manyfold greater than those in diabetic dogs showing glomerulopathy. In rats, but not in dogs, dietary protein content has been identified as a factor in the development of glomerulosclerosis and decreased renal function. Glomerulosclerosis can be induced in normal rats by feeding a high-protein diet (22) and can be inhibited in diabetic rats by severe protein restriction (23). However, in dogs with 75% nephrectomy, consumption of a high-protein diet (56% protein) for several years has been reported not to reproducibly affect renal morphology or to impair renal function (24). Diabetic dogs in our study consumed twice as much food as the galactosemics, but there is little reason to speculate that the associated difference in protein intake contributed to the observed difference in glomerular pathology between the two groups. The glomeruli from galactose-fed dogs were not entirely normal. Basement membrane thickness of glomerular capillaries and the quantity of immunohistochemically demonstrable plasma proteins within the glomeruli were supranormal. The glomerular basement membrane thickening observed in the galactosemic dogs may be an early manifestation of a slowly developing glomerulopathy. Alternatively, the pathogenesis of the basement membrane thickening might be different from that of mesangial expansion, glomerular obliteration, and renal hypertrophy in diabetes. Basement membrane thickening in retinal capillaries of rats reportedly can be inhibited by pharmacologic inhibitors of polyol production (25,26), but effects of such inhibitors on renal histopathology have yet to be described. Treatment of diabetic subjects with pharmacologic inhibitors of polyol production should be expected to influence some renal lesions less than others, because in the experiment reported herein, excessive polyol production was associated with glomerular basement membrane thickening and plasma protein deposition but not necessarily with mesangial expansion, glomerular obliteration, or renal hypertrophy. Accumulation of plasma proteins within glomerular basement membrane tends to be excessive in diabetes and, as reported herein, in experimental galactosemia. The accumulation of plasma proteins within glomeruli was associated with basement membrane thickening both in galactosemia and diabetes and, like basement membrane thickening, appears to be secondary to hyperglycemia. How these proteins accumulate or if such accumulation plays a role in the development of diabetic glomerulopathy is unclear. Nevertheless, the absence of mesangial expansion in galactosemic dogs showing accumulation of plasma proteins indicates that such deposits do not necessarily result in mesangial expansion. ACKNOWLEDGMENTS We are grateful to M. Garment, M. Larson, and H. Wabers for technical assistance. This work was supported in part by PHS research Grant EY from the National Eye Institute, by a grant from the Juvenile Diabetes Foundation, and by a Lions Clubs International Research and Development Award from the American Diabetes Association. REFERENCES 1. Rasch R: Prevention of diabetic glomerulopathy in streptozotocin diabetic rats by insulin treatment. 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