Effects of Pancreatic Islet Transplantation on the Increased Urinary Albumin Excretion Rates in Intact and Uninephrectomized Rats with Diabetes Mellitus S. Michael Mauer, M.D., David M. Brown, M.D., Arthur J. Matas, M.D., and Michael W. Steffes, M.D., Ph.D., Minneapolis SUMMARY The effects of reduced renal mass and of islet transplantation on urinary albumin excretion (UAE) rates in diabetic rats were examined. Increased UAE rates were noted two months after the induction of streptozotocin diabetes in 100-gm. inbred Lewis rats. Over the subsequent seven months, UAE rates remained constant in controls but were increasing in diabetic rats at the end of this time. Uninephrectomized nondiabetic rats had increasing UAE with time as compared with intact controls. Uninephrectomized diabetic rats had greater UAE than their respective controls, and the magnitude of this difference increased over the nine-month period of these studies. Uninephrectomized and intact rats, diabetic for seven months, were cured of the diabetic state by neonatal pancreatic tissue administered via the portal vein. Within one month of transplantation, UAE values decreased in these animals
as compared with their pretransplant values and, within two months, were no longer different from values in their respective nondiabetic controls. In contrast, untreated uninephrectomized and intact diabetic rats increased their UAE over these same two months. At two months posttransplant, glomerular mesangial thickening was decreased in transplanted uninephrectomized and intact diabetic rats when compared with their respective untreated diabetic littermates. Thus, the albuminuria of diabetes in rats is reversed by pancreatic islet transplantation. It is unclear whether this reversal is primarily due to improvement in functional or structural abnormalities in glomeruli of diabetic rats. DIABETES 27:959-64, September, 1978.
The pathogenesis of increased urinary albumin excretion in experimental and human diabetes mellitus is unclear. The clinical course of juvenile-onset diabetic patients dying of diabetic nephropathy has been characterized by the development of persistent albuminuria (more than 20 mg./lOO ml. in random urine specimens) after a mean duration of diabetes of 17.3 years.1 This was followed, on the average, by early renal failure within two years and by death from renal failure within four years.1 In another study, 2 although the duration of proteinuria before the onset of early renal failure was variable, the frequency of azotemia approached 50 per cent within five years of the onset of proteinuria; of 25 patients developing
azotemia, 23 were dead within two years. Thus, persistent overt proteinuria generally occurs late in the evolution of diabetic glomerulosclerosis and is associated with advanced diffuse or nodular glomerular lesions and hyaline arteriolar degenerative changes. 3 " 10 Using a sensitive radioimmunoassay11 that measures albuminuria not detected by standard techniques, Mogensen showed that juvenile-onset diabetic patients usually have normal basal urinary albumin excretion rates despite diabetes of several years' duration. However, levels of exercise insufficient to increase urinary albumin excretion rates in normal subjects did increase these rates in diabetic patients with duration of disease of four to 16 years. 13 Most of these patients would have been expected to manifest glomerular basement membrane (GBM) and mesangial thickening. 14 Further, Mogensen12 showed that newly diagnosed juvenile diabetics in poor control had elevated urinary albumin excretion rates that became normal when strict metabolic control was instituted.
From the Departments of Pediatrics, Laboratory Medicine and Pathology, and Surgery, University of Minnesota Medical School, Minneapolis. Address reprint requests to S. Michael Mauer, M.D., Department of Pediatrics, Box 491—Mayo, University of Minnesota, Minneapolis, Minnesota 55455Accepted for publication April 10, 1978. SEPTEMBER, 1978
959
ALBUMINURIA IN DIABETIC RATS
Many of these patients with short duration of clinical diabetes would not have been expected to have measurable GBM thickening. 14 Further, unlike the patients with diabetes of longer duration but in good metabolic control, the newly diagnosed patients in poor control probably had a component of decreased tubular protein reabsorption. 15 Few careful studies of urinary albumin excretion have been performed in animal models of diabetes. Mice with genetic diabetes may develop proteinuria before development of the structural glomerular alterations. 16 Weil et al., 1 7 measuring a urinary glycoprotein, 18 found increased excretion rates within four weeks of onset of streptozotocin-induced diabetes that steadily increased over the next eight months in contrast with the lower and stable rates in control animals. However, the relationship of this protein to glomerular permeability is uncertain. Hagg, using a modification of the Lowry method, noted increased urinary albumin content in diabetic rats at one year but not at two weeks after alloxan injection. 19 Bloodworth 20 found qualitatively increased urinary protein concentrations in long-term alloxan-diabetic dogs with glomerular lesions remarkably similar to those seen in man. We have previously commented on the similarities in the glomerular changes of long-term diabetes in rats and man, 2 1 ' 2 2 including mesangial matrix 21 and GBM thickening, 19 glomerular hyaline nodular deposits containing immunoglobulins and complement capable of fixing heterologous complement, 21 and increased mesangial staining for smooth muscle proteins. 23 In addition we have shown that unilateral nephrectomy accelerates the development of glomerulopathy in streptozotocin-diabetic rats. 2 4 Further, we have shown improvement in diabetic glomerulopathy in rats cured of the diabetic state by pancreatic islet transplantation. 2 5 ' 2 6 The present studies demonstrate that increased urinary albumin excretion (UAE) rates found in intact and uninephrectomized rats with long-term diabetes return to control values after islet transplantation. MATERIALS AND METHODS Animals. Highly inbred Lewis male rats (Simonson Labs, Gilroy, California) weighing roughly 100 gm. (six weeks of age) were made diabetic with streptozotocin* as previously described. 27 Age-matched littermates served as controls. Induction of the diabetic state was confirmed by the development of persis*Kindly supplied by Upjohn Co., Kalamazoo, Michigan.
960
tent, nonfasting, plasma glucose concentrations in excess of 400 mg. per deciliter. Two weeks later, one half of the diabetic and control groups of animals underwent unilateral nephrectomy. Seven months after the time of diabetes' induction, some of the uninephrectomized and intact diabetic rats were selected at random for pancreatic islet tissue transplantation using the method of Matas et al. 28 Thus, the following groups were available for study: (a) diabetic, nontransplanted (D)—29 rats with diabetes up to four months and 16 rats thereafter; (b) unilaterally nephrectomized diabetic, nontransplanted (UND)—30 rats up to four months and nine rats thereafter; (c) diabetic, transplanted (D-T)—nine rats; (d) unilaterally nephrectomized diabetic, transplanted (UND-T)—eight rats; (e) nondiabetic controls (C)—13 rats; and (f) unilaterally nephrectomized nondiabetic controls (UNC)—13 rats. All animals were maintained on Purina rat chow and on water ad libitum throughout the experiment. Urinary Albumin Excretion Urine was collected from all animals by placing them in metabolic cages without food but with free access to water for 18 hours; the cages were rinsed with water, and the albumin content from this total collection was determined by single radial immunodiffusion using monospecific rabbit antibody to rat albumin. 29 - 30 Each urine sample was studied in duplicate along with a pure rat albumin standard. This standard was rat albumin (Melroy Labs, Springfield, Virginia), which was further purified over a 0.5 M Biogel (Bio-Rad Labs, Richmond, California) column followed by an ion-exchange salt gradient column using DE23 (Reeve-Angel, Clifton, N.J.). This final material was shown to have a single line on Ouchterlony plates and in immunoelectrophoresis against rabbit anti—whole rat antiserum. The standard deviation between duplicate samples, based on range statistics, 31 was 1.5 /Ag. per milliliter of urine, resulting in a coefficient of variation of 2.7 per cent using the median standard; the coefficient of variation for control samples tested on different plates was 12 per cent. The sensitivity of the assay was 14 fig. per milliliter. Results are presented as milligrams urinary albumin per 24 hours per 100 gm. body weight expressed as means ± S.E.M. for each group. Other Methods
Plasma glucose concentrations were determined in nonfasting animals using the glucose oxidase method on a glucose analyzer (Beckman Instruments, Fullerton, California). Tissue was prepared for light microsD1ABETES, VOL. 2 7 , NO. 9
S. MICHAEL MAUER, M.D., AND ASSOCIATES
copy, and PAS-stained sections were examined and graded as previously described;26 results were expressed as means ± S.E.M. for each group for the index of mesangial thickening. Statistical methods included paired and unpaired Student's /-tests. Results of p-values represent unpaired /-tests unless stated otherwise.
10.0-
RESULTS D rats had mean UAE rates three to four times greater than C at two, seven,' and nine months after the induction of diabetes (p-values < 0 . 0 0 1 , < 0 . 0 0 1 , and < 0 . 0 0 5 , respectively, figure 1). The mean UAE £ E F CT of C rats remained constant over this time while that of D rats was increased at the nine-month time period, although significantly so only when compared with the four-month (p < 0.05) and the seven-month (p < 0.05) levels (paired /-test). UNC rats at six weeks after uninephrectomy (i.e., two-month time period, figure 1) had significantly higher UAE rates than did C animals (p < 0.01). Further, unlike C animals, UNC rats had increasing UAE rates over the next seven months (figure 1 and table 1) so that by nine months their mean UAE was almost 30 times the C value (p < 0.01). UND ani6 8 10 mals (figure 1 and table 1) had mean UAE rates about Months of Diabetes three to four times those of UNC animals after two (p FIG. 1. Urinary albumin excretion in control, diabetic, unilaterally < 0.001), four (p < 0.001), seven (p < 0.05), and nephrectomized nondiabetic, and unilaterally nephrecnine (p < 0.05) months of diabetes, respectively. As tomized diabetic rats. In this and the subsequent figures, the S.E.M.'s of both the UNC and UND animals' rates diabetic animals were given streptozotocin at time 0. On this time scale, uninephrectomy was carried out two weeks of UAE indicate (figure 1), considerable variation later. The two- and four-month time periods include all among animals occurred at the later time periods in animals entering the study. Data from those uninephrectomized and intact diabetic animals not receiving panthese groups. creatic islet tissue are presented at seven and nine Seven months after induction of diabetes, pancremonths. atic islet transplantation was carried out in nine D and eight UND animals. Before transplantation, nonfastUAE rate in D-T rats was not different from that in C ing plasma glucose concentrations exceeded 500 mg. rats, while both were lower than in D animals (p < per deciliter in all these animals. These levels fell to 0.005 for both comparisons). roughly 250 mg. per deciliter within two weeks after At the time of transplantation the mean UAE rate transplantation, reached the normal range within four TABLE 1 to six weeks, and remained normal thereafter. Results of statistical analysis of increasing UAE rate At the time of transplantation the mean UAE rate with time in UNC and UND rats of D-T rats was higher than that of D rats, although this difference did not reach statistical significance Months of diabetes (figure 2). However, the means of both groups were 2 mo. 7 mo. 4 mo. vs. 4 mo. vs. 7 mo. vs. 9 mo. higher than those of C (p < 0.05). While, as noted p < 0.005 p < 0.05 P < 0.01 above, UAE increased in D animals over the sub- UND Paired /-test p < 0.05 Unpaired r-test p < 0.05 P< 0.025 sequent two months, D-T animals had a marked reUNC Paired r-test N.S. p < 0.01 P < 0.005 duction in UAE during this same time. Within one Unpaired r-test N.S. p < 0.025 P < 0.025 month of transplantation the mean UAE rate in D-T UAE, urinary albumin excretion; UND, unilaterally nephrectomized animals was less than their pretransplantation rate (p diabetic; UNC, unilaterally nephrectomized nondiabetic; N.S., not statistically significant, i.e., p < 0.05. < 0.05). Two months after transplantation the mean
i
SEPTEMBER, 1978
961
ALBUMINURIA IN DIABETIC RATS
of UND and UND-T animals were similar. Over the 12 Mean t SEM next two months, UAE rose markedly in UND animals compared with their seven-month values (figures 1 and 3 and table 1). In contrast (figure 3), UAE rate in UND-T rats fell within two weeks of trasplantation 10 compared with their pretransplant values (p < 0.05; UNILATERAL NEPHRECTOMY O) AND DIABETES paired /-test). Two months after transplantation UND-T rats had UAE rates that did not differ significantly from UNC animals but that were less than one-seventh those of UND rats (p < 0.02). The index of mesangial thickening (IMT) after E ra seven months of diabetes was 1.7 ± 0.1 in D rats as compared with 2.6 ± 0.01 in UND rats (p < < 7TRANSPLANTED 0.0005). Both D and UND animals had IMTs greater UNILATERAL NEPHRECTOMY AND DIABETES than their respective nondiabetic controls at this time (p < 0.0005 for each comparison). Two months after transplantation IMTs were 0.7 ± 0.2 in D-T and 1.1 ± 0.2 in UND-T animals, both being lower than their corresponding pretransplant values (p < 0.0005 and < 0.005, respectively). However, at this time UNILATERAL IMTs in D-T and UND-T rats remained higher than NEPHRECTOMY their corresponding nondiabetic littermates (p < 4 6 8 10 o 0.005 and < 0.0005, respectively). In the sevenMonths of Diabetes month compared with the nine-month time period FIG. 3. Urinary albumin excretion in unilaterally nephrectomized the IMT in D animals increased slightly to 2.0 ± nondiabetic, nontransplanted uninephrectomized diabetic, and uninephrectomized transplanted diabetic rats. At 0.08 (p < 0.05) while in UND rats no change in IMT transplantation (seven months) the uninephrectomized occurred. At the nine-month time period (two months transplanted animals were separated from the nontransplanted uninephrectomized diabetic group, and both groups were studied separately at that time and thereafter.
Mean i SEM
posttransplant) IMTs were lower in D-T and UND-T as compared with D and UND rats, respectively (p < 0.0005 for each comparison).
08
O.6
TRANSPLANTED DIABETES^
DISCUSSION r
c E
Eg 04
DIABETES
3 Q *
Q2
CONTROL
11
5 4
6
8
TO
Months of Diabetes FIG. 2.
962
Urinary albumin excretion in control, nontransplanted diabetic, and transplanted diabetic rats. At transplantation (seven months) the transplanted diabetic animals were separated from the nontransplanted diabetic group, and both groups were studied separately at that time and thereafter. Note change in the scale of the ordinate when compared with figures 1 and 3.
These studies confirm that proteinuria occurs in rats with experimental diabetes mellitus. In D animals UAE, higher than C throughout the study, increased further after seven months of diabetes. In contrast both UND and UNC rats had rising UAE rates with time, and the magnitude of the differences between these two groups increased progressively. Reversal of the diabetic state by pancreatic islet transplantation caused a return of UAE in both D-T and UND-T animals to their respective control levels within two months. Although paralleling the return to normoglycemia, the decline in UAE of D-T and UND-T rats lagged about two to three weeks behind the decline in plasma glucose values induced by transplantation. It is known that the major light, immunofluorescent, and electron microscopic alterations of diabetic DIABETES, VOL. 27, NO. 9
S. MICHAEL MAUER, M.D., A N D ASSOCIATES
glomerulopathy in rats are secondary to the diabetic state. Progressive mesangial thickening and mesangial deposition of immunoglobulins and complement 21 can be reversed after transplantation of a kidney from a diabetic to a normal host 27 or after successful pancreatic islet transplantation. 25 - 26 Further, GBM thickening in diabetic rats can be retarded in its development by instituting measures to lower blood sugar, including insulin administration and reduction in carbohydrate intake. 3 2 ' 3 3 Alterations in glomerular function in the earlier stages of diabetic nephropathy in man have been documented. 15 Studies of graded dextran polymer clearances in newly diagnosed or longer term diabetic patients have not indicated alterations in glomerular capillary filtration properties. 1 5 ' 3 4 The increased glomerular filtration rate, filtration fraction, and UAE with exercise in early diabetes have been interpreted as resulting from increased glomerular-filtering area and glomerular capillary pressures rather than from structural abnormalities of the glomerular capillary filter system. Azar* has documented elevated glomerular capillary pressures in diabetic rats. We have shown that unilateral nephrectomy accelerates the development of diabetic nephropathy in rats, 24 perhaps on the basis of increased glomerular blood flow and capillary pressures. 34 Thus it is possible that the exercise-induced albuminuria in diabetic man and the increased UAE in diabetic rats result from functional glomerular alterations and that unilateral nephrectomy, by accentuating these alterations, results in even greater urinary albumin loss. Although indirect evidence suggests that the mechanism for tubular reabsorption of protein in rats normally is not operating at a maximum, 36 ' 37 direct Bowman's capsule or proximal tubule micropuncture studies have shown that even minimally increased glomerular permeability to albumin results in increased quantities of albumin in the final urine. 3 8 ' 3 9 It is possible that exercise is required to unmask abnormalities in transglomerular capillary passage of albumin in man with well controlled diabetes 13 because a tubular threshold for albumin reabsorption must be surpassed. Diabetic patients in poor metabolic control, a situation more analogous to the present studies in rats, do have increased albuminuria at rest, apparently due to increased quantities of albumin in glomerular filtrate and, to a lesser extent, decreased tubular reabsorptive capacity for proteins. 15 ' 40 This albuminuria, associated with increased kidney size, *Azar, S., personal communication with me (SMM). SEPTEMBER, 1978
glomerular filtration rate, and filtration fraction, is, at least in part, reversible by the establishment of good metabolic control. 15 ' 40 Thus, the reversal of increased UAE by the achievement of normoglycemia after islet transplantation may be due to reversal of functional changes in glomerular hemodynamics. However, improvement in certain aspects of the glomerular pathology of diabetes in the rat has been documented to occur as early as two weeks after pancreas transplantation 26 and was clearly evident two months after transplantation in the present experiments. Thus, we cannot exclude the possibility that structural changes in the glomerular capillary filter system contribute to increased albuminuria in diabetic rats and that reversal of these changes by pancreas transplantation eliminates the increase. At this time it is not clear whether the increased UAE rate noted in our diabetic animals is analagous to albuminuria in the early stages of diabetic nephropathy in man or whether this increase more closely resembles the proteinuria that presages early renal failure in humans with severe diabetic glomerulopathy. 1 ' 2 Studies of differential renal protein clearances in diabetic patients have demonstrated differences in urinary IgG-transferrin clearance ratios in the early as compared with the late stages of diabetic nephropathy. 41 Studies along these and other lines may help to further elucidate the phenomena reported herein. ACKNOWLEDGMENTS We thank Ms. C. Swope, S. Sandberg, M. Stahlman, M. Rudquist, and Mr. J. Basgen for their excellent technical assistance. The rabbit anti-rat albumin antibody was generously supplied by Dr. J. R. Hoyer, Boston. We appreciate the secretarial and editorial assistance of Ms. Trudy Bensfield. Susan Perry drew the figures. We thank Esther Freier and Lorraine Gonyea-Stewart for their help with the statistical validation of the urinary albumin methodology. This work was supported by N . I . H . grants AM 17697 and HL-06314. REFERENCES ^ u s s m a n , M. J., Goldstein, H. H . , and Gleason, R. E.: The clinical course of diabetic nephropathy. JAMA 236:1861-63, 1976. 2 Knowles, H. C , Jr.: The magnitude of the renal failure problem in diabetic patients. Kidney Int. 6 (Suppl. l):2-7. 1974. 3 Rogers, J., Robbins, S. L., and Jeghers, H.G Intercapillary glomerulosclerosis: a clinical and pathologic study. II. A clinical study of 100 anatomically proven cases. Am. J. Med. 72:692-99, 1952. 963
ALBUMINURIA IN DIABETIC RATS 4
Bell, E. T.: Renal vascular .disease in diabetes mellitus. Diabetes 2:376-89, 1953. 5 Taft, H. P., Finckh, E. S., and Joske, R. A.: A biopsy study of the kidney in diabetes mellitus. Australas. Ann. Med. 3:189201, 1954. 6 Lambie, A. T., and MacFarlane, A.: A clinico-pathologic study of diabetic glomerulosclerosis. Q. J. Med. 24:125-42, 1955. 7 Gellman, D, D . , Pirani, C. L., Soothill, J. F., Muehrcke, R. C , and Kark, R. M.: Diabetic nephropathy: a clinical and pathological study based on renal biopsies. Medicine (Baltimore) 38:321-67, 1959. 8 Hennigar, G. F., Cohen, R. J., and Katz, H. P.: Nodular glomerulosclerosis: clinico-pathological correlation of 40 advanced cases. Am. J. Med. Sci. 42:89-99, 1961. 9 Thomsen, A. C : The Kidney in Diabetes Mellitus. Copenhagen, Munksgaard, 1965, pp. 208-15. 10 Yamauchi, Y., Suzuki, J., Ohneda, A., Yamagata, S., and Goto, Y.: Onset and progress of diabetic glomerulosclerosis. Toh o k u j . Exp. Med. 209:385-406, 1973. n Miles, D. W . , Mogensen, C. E., and Gunderson, H. J. G.: Radioimmunoassay for urinary albumin using a single antibody. Scand. J. Clin. Lab. Invest. 26:5-11, 1970. 12 Mogensen, C. E.: Urinary albumin excretion in early and long-term juvenile diabetes. Scand. J. Clin. Lab. Invest. 28:183-93, 1971. 13 Mogensen, C. E., and Vittinghus, E.: Urinary albumin excretion during exercise in juvenile diabetes: a provocative test for early abnormalities. Scand. J. Clin. Lab. Invest. 3.5:295-300, 1975. 14 0sterby, R.: Early phases in the development of diabetic glomerulopathy. Acta Med. Scand. Suppl. .574:1-80, 1975. 15 Mogensen, C. E.: Renal function changes in diabetes. Diabetes 25 (Suppl. 2):872-79, 1976. 16 Galaske, R. G., Gartner, K., and Stolte, H.: Functional and morphological study on the onset of proteinuria in experimental diabetes mellitus. In Contr. to Nephrology, Vol. 1. Eisenbach, G. M., Brod, J., and Stolte, H., Editors. Basel, Karger, 1975, pp. 69-79. 17 Weil, R., I l l , Nozawa, M., Koss, M . , Weber, C , Reemtsma, K., and Mclntosh, R.: The kidney in streptozotocin diabetic rats. Morphologic, ultrastructural and functional studies. Arch. Pathol. Lab. Med. 200:37-49, 1976. 18 Griswold, W. R., and Mclntosh, R. M.: Increased urinary excretion of a basement membrane-like glycoprotein in acute uranium nephropathy. Experentia 29:575-76, 1973. 19 Hagg, E.: Renal lesions in rats with long-term alloxan diabetes. Acta Pathol. Microbiol. Scand. 82:199-209, 1974. 20 Bloodworth, J. M. B . , J r . : Experimental diabetic glomerulosclerosis. II. The dog. Arch. Pathol. 79:113-25, 1965. 21 Mauer, S. M., Michael, A. F., Fish, A. J., and Brown, D. M.: Spontaneous immunoglobulin and complement deposition in glomeruli of diabetic rats. Lab. Invest. 27:488-94, 1972. 22 Mauer, S. M., Steffes, M. W . , Michael, A. F., and Brown, D. M.: Studies of diabetic nephropathy in animals and man. Diabetes 25 (Suppl. 2):850-57, 1976. 23 Scheinman, J. I., Steffes, M. W . , Brown, D. M., and Mauer, S. M.: The immunohistopathology of glomerular antigens. III. Increased mesangial actomyosin in experimental diabetes in the rat. Diabetes 27:632-37, 1978. "Steffes, M. W . , Brown, D. M., and Mauer, S. M.: Diabetic glomerulopathy following unilateral nephrectomy in the rat.
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Diabetes 27:35-41, 1978. 25 Mauer, S. M., Sutherland, D. E. R., Steffes, M. W . , Leonard, R. J., Najarian, J. S., Michael, A. F., and Brown, D. M.: Pancreatic islet transplantation: effects on the glomerular lesions of experimental diabetes in the rat. Diabetes 23:748-53, 1974. 26 Mauer, S. M., Steffes, M. W . , Sutherland, D. E. R., Najarian, J. S., Michael, A. F., and Brown, D. M.: Studies of the rate of regression of the glomerular lesions in diabetic rats treated with pancreatic islet transplantation. Diabetes 24:280-85, 1975. "Lee, C. S., Mauer, S. M., Brown, D. M., Sutherland, D. E. R., Michael, A. F., and Najarian, J. S.: Renal transplantation in diabetes mellitus in rats. J. Exp. Med. 239:793-800, 1974. 28 Matas, A. J., Payne, W. D., Grotting, J. C , Sutherland, D. E. R., Steffes, M. W . , Hertel, B. F., and Najarian, J. S.: Portal versus systemic transplantation of dispersed neonatal pancreas. Transplantation 24:333-37, 1977. 29 Mancini, G., Carbonara, A. O., and Heneman, J. R.: Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 2:235-54, 1965. 30 Hoyer, J. R., Mauer, S. M., and Michael, A. F.: Unilateral renal disease in the rat. I. Clinical, morphologic and glomerular mesangial functional features of the experimental model produced by renal perfusion with aminonucleoside. J. Lab. Clin. Med. 85:756-68, 1975. 31 ASTM Manual on Quality Control of Materials. Philadelphia, American Society for Testing Materials, I960, p. 63. 32 Hagg, E.: Influence of insulin treatment on glomerular changes in rats with long-term alloxan diabetes. Acta Pathol. Microbiol. Scand. 32:228-34, 1974. 33 Fox, C. J., Darby, S. C , Ireland, J. T., and Sorksen, P. D.: Blood glucose control and glomerular capillary basement membrane thickening in experimental diabetes. Br. Med. J. 2:605-07, 1977. 34 Mogensen, C. E.: Kidney function and glomerular permeability to macro-molecules in early juvenile diabetes. Scand. J. Clin. Lab. Invest. 28:79-90, 1971. 35 Azar, S., Johnson, M. A., Hertell, B., and Tobian, L.: Single-nephron pressures, flows and resistances in hypertension with nephrosclerosis. Kidney Int. 22:28-40, 1977. 36 Mendel, D.: Tubular reabsorption of protein in rats with experimental proteinuria. J. Physiol. (London) 2.56:44-54, 1961. 37 Bordeau, J. E., and Carone, F. A.: Protein handling by the renal tubule. Nephron 23:22-34, 1974. 38 Oken, D. E., Cotes, S. C , and Mende, C. W.: Micropuncture study of tubular transport of albumin in rats with aminonucleoside nephrosis. Kidney Int. 22:291-304, 1972. 39 Stolte, H., Kuhn, K., Galaske, R., Alt, J., Baldamus, C , Eisenbach, G. M., and Sterzel, R. B.: Function and structure following glomerular permeability changes in anti-GBM nephritis and aminonucleoside nephrosis. In Contr. to Nephrology, Vol. II. Sterzel, R. B., Thomson, D., and Brod, J., Editors. Basel, Karger, 1976, p. 114. 40 Parving, H. H . , Noer, I., Deckert, T., Evrin, P. E., Nielsen, S. L., Lynsg0e, J., Mogensen, C. E., R0rth, M., Svendsen, P. A., Trap-Jensen, J., and Lassen, N . A.: The effect of metabolic regulation on microvascular permeability to small and large molecules in short-term juvenile diabetes. Diabetologia 22:161-66, 1976. 41 Jerums, G., Post, R. S., Miller, M., and Barzellato, E.: Differential renal protein clearance in diabetes. Diabetes 22:10410, 1973. DIABETES, VOL. 2 7 , NO. 9
as compared with their pretransplant values and, within two months, were no longer different from values in their respective nondiabetic controls. In contrast, untreated uninephrectomized and intact diabetic rats increased their UAE over these same two months. At two months posttransplant, glomerular mesangial thickening was decreased in transplanted uninephrectomized and intact diabetic rats when compared with their respective untreated diabetic littermates. Thus, the albuminuria of diabetes in rats is reversed by pancreatic islet transplantation. It is unclear whether this reversal is primarily due to improvement in functional or structural abnormalities in glomeruli of diabetic rats. DIABETES 27:959-64, September, 1978.
The pathogenesis of increased urinary albumin excretion in experimental and human diabetes mellitus is unclear. The clinical course of juvenile-onset diabetic patients dying of diabetic nephropathy has been characterized by the development of persistent albuminuria (more than 20 mg./lOO ml. in random urine specimens) after a mean duration of diabetes of 17.3 years.1 This was followed, on the average, by early renal failure within two years and by death from renal failure within four years.1 In another study, 2 although the duration of proteinuria before the onset of early renal failure was variable, the frequency of azotemia approached 50 per cent within five years of the onset of proteinuria; of 25 patients developing
azotemia, 23 were dead within two years. Thus, persistent overt proteinuria generally occurs late in the evolution of diabetic glomerulosclerosis and is associated with advanced diffuse or nodular glomerular lesions and hyaline arteriolar degenerative changes. 3 " 10 Using a sensitive radioimmunoassay11 that measures albuminuria not detected by standard techniques, Mogensen showed that juvenile-onset diabetic patients usually have normal basal urinary albumin excretion rates despite diabetes of several years' duration. However, levels of exercise insufficient to increase urinary albumin excretion rates in normal subjects did increase these rates in diabetic patients with duration of disease of four to 16 years. 13 Most of these patients would have been expected to manifest glomerular basement membrane (GBM) and mesangial thickening. 14 Further, Mogensen12 showed that newly diagnosed juvenile diabetics in poor control had elevated urinary albumin excretion rates that became normal when strict metabolic control was instituted.
From the Departments of Pediatrics, Laboratory Medicine and Pathology, and Surgery, University of Minnesota Medical School, Minneapolis. Address reprint requests to S. Michael Mauer, M.D., Department of Pediatrics, Box 491—Mayo, University of Minnesota, Minneapolis, Minnesota 55455Accepted for publication April 10, 1978. SEPTEMBER, 1978
959
ALBUMINURIA IN DIABETIC RATS
Many of these patients with short duration of clinical diabetes would not have been expected to have measurable GBM thickening. 14 Further, unlike the patients with diabetes of longer duration but in good metabolic control, the newly diagnosed patients in poor control probably had a component of decreased tubular protein reabsorption. 15 Few careful studies of urinary albumin excretion have been performed in animal models of diabetes. Mice with genetic diabetes may develop proteinuria before development of the structural glomerular alterations. 16 Weil et al., 1 7 measuring a urinary glycoprotein, 18 found increased excretion rates within four weeks of onset of streptozotocin-induced diabetes that steadily increased over the next eight months in contrast with the lower and stable rates in control animals. However, the relationship of this protein to glomerular permeability is uncertain. Hagg, using a modification of the Lowry method, noted increased urinary albumin content in diabetic rats at one year but not at two weeks after alloxan injection. 19 Bloodworth 20 found qualitatively increased urinary protein concentrations in long-term alloxan-diabetic dogs with glomerular lesions remarkably similar to those seen in man. We have previously commented on the similarities in the glomerular changes of long-term diabetes in rats and man, 2 1 ' 2 2 including mesangial matrix 21 and GBM thickening, 19 glomerular hyaline nodular deposits containing immunoglobulins and complement capable of fixing heterologous complement, 21 and increased mesangial staining for smooth muscle proteins. 23 In addition we have shown that unilateral nephrectomy accelerates the development of glomerulopathy in streptozotocin-diabetic rats. 2 4 Further, we have shown improvement in diabetic glomerulopathy in rats cured of the diabetic state by pancreatic islet transplantation. 2 5 ' 2 6 The present studies demonstrate that increased urinary albumin excretion (UAE) rates found in intact and uninephrectomized rats with long-term diabetes return to control values after islet transplantation. MATERIALS AND METHODS Animals. Highly inbred Lewis male rats (Simonson Labs, Gilroy, California) weighing roughly 100 gm. (six weeks of age) were made diabetic with streptozotocin* as previously described. 27 Age-matched littermates served as controls. Induction of the diabetic state was confirmed by the development of persis*Kindly supplied by Upjohn Co., Kalamazoo, Michigan.
960
tent, nonfasting, plasma glucose concentrations in excess of 400 mg. per deciliter. Two weeks later, one half of the diabetic and control groups of animals underwent unilateral nephrectomy. Seven months after the time of diabetes' induction, some of the uninephrectomized and intact diabetic rats were selected at random for pancreatic islet tissue transplantation using the method of Matas et al. 28 Thus, the following groups were available for study: (a) diabetic, nontransplanted (D)—29 rats with diabetes up to four months and 16 rats thereafter; (b) unilaterally nephrectomized diabetic, nontransplanted (UND)—30 rats up to four months and nine rats thereafter; (c) diabetic, transplanted (D-T)—nine rats; (d) unilaterally nephrectomized diabetic, transplanted (UND-T)—eight rats; (e) nondiabetic controls (C)—13 rats; and (f) unilaterally nephrectomized nondiabetic controls (UNC)—13 rats. All animals were maintained on Purina rat chow and on water ad libitum throughout the experiment. Urinary Albumin Excretion Urine was collected from all animals by placing them in metabolic cages without food but with free access to water for 18 hours; the cages were rinsed with water, and the albumin content from this total collection was determined by single radial immunodiffusion using monospecific rabbit antibody to rat albumin. 29 - 30 Each urine sample was studied in duplicate along with a pure rat albumin standard. This standard was rat albumin (Melroy Labs, Springfield, Virginia), which was further purified over a 0.5 M Biogel (Bio-Rad Labs, Richmond, California) column followed by an ion-exchange salt gradient column using DE23 (Reeve-Angel, Clifton, N.J.). This final material was shown to have a single line on Ouchterlony plates and in immunoelectrophoresis against rabbit anti—whole rat antiserum. The standard deviation between duplicate samples, based on range statistics, 31 was 1.5 /Ag. per milliliter of urine, resulting in a coefficient of variation of 2.7 per cent using the median standard; the coefficient of variation for control samples tested on different plates was 12 per cent. The sensitivity of the assay was 14 fig. per milliliter. Results are presented as milligrams urinary albumin per 24 hours per 100 gm. body weight expressed as means ± S.E.M. for each group. Other Methods
Plasma glucose concentrations were determined in nonfasting animals using the glucose oxidase method on a glucose analyzer (Beckman Instruments, Fullerton, California). Tissue was prepared for light microsD1ABETES, VOL. 2 7 , NO. 9
S. MICHAEL MAUER, M.D., AND ASSOCIATES
copy, and PAS-stained sections were examined and graded as previously described;26 results were expressed as means ± S.E.M. for each group for the index of mesangial thickening. Statistical methods included paired and unpaired Student's /-tests. Results of p-values represent unpaired /-tests unless stated otherwise.
10.0-
RESULTS D rats had mean UAE rates three to four times greater than C at two, seven,' and nine months after the induction of diabetes (p-values < 0 . 0 0 1 , < 0 . 0 0 1 , and < 0 . 0 0 5 , respectively, figure 1). The mean UAE £ E F CT of C rats remained constant over this time while that of D rats was increased at the nine-month time period, although significantly so only when compared with the four-month (p < 0.05) and the seven-month (p < 0.05) levels (paired /-test). UNC rats at six weeks after uninephrectomy (i.e., two-month time period, figure 1) had significantly higher UAE rates than did C animals (p < 0.01). Further, unlike C animals, UNC rats had increasing UAE rates over the next seven months (figure 1 and table 1) so that by nine months their mean UAE was almost 30 times the C value (p < 0.01). UND ani6 8 10 mals (figure 1 and table 1) had mean UAE rates about Months of Diabetes three to four times those of UNC animals after two (p FIG. 1. Urinary albumin excretion in control, diabetic, unilaterally < 0.001), four (p < 0.001), seven (p < 0.05), and nephrectomized nondiabetic, and unilaterally nephrecnine (p < 0.05) months of diabetes, respectively. As tomized diabetic rats. In this and the subsequent figures, the S.E.M.'s of both the UNC and UND animals' rates diabetic animals were given streptozotocin at time 0. On this time scale, uninephrectomy was carried out two weeks of UAE indicate (figure 1), considerable variation later. The two- and four-month time periods include all among animals occurred at the later time periods in animals entering the study. Data from those uninephrectomized and intact diabetic animals not receiving panthese groups. creatic islet tissue are presented at seven and nine Seven months after induction of diabetes, pancremonths. atic islet transplantation was carried out in nine D and eight UND animals. Before transplantation, nonfastUAE rate in D-T rats was not different from that in C ing plasma glucose concentrations exceeded 500 mg. rats, while both were lower than in D animals (p < per deciliter in all these animals. These levels fell to 0.005 for both comparisons). roughly 250 mg. per deciliter within two weeks after At the time of transplantation the mean UAE rate transplantation, reached the normal range within four TABLE 1 to six weeks, and remained normal thereafter. Results of statistical analysis of increasing UAE rate At the time of transplantation the mean UAE rate with time in UNC and UND rats of D-T rats was higher than that of D rats, although this difference did not reach statistical significance Months of diabetes (figure 2). However, the means of both groups were 2 mo. 7 mo. 4 mo. vs. 4 mo. vs. 7 mo. vs. 9 mo. higher than those of C (p < 0.05). While, as noted p < 0.005 p < 0.05 P < 0.01 above, UAE increased in D animals over the sub- UND Paired /-test p < 0.05 Unpaired r-test p < 0.05 P< 0.025 sequent two months, D-T animals had a marked reUNC Paired r-test N.S. p < 0.01 P < 0.005 duction in UAE during this same time. Within one Unpaired r-test N.S. p < 0.025 P < 0.025 month of transplantation the mean UAE rate in D-T UAE, urinary albumin excretion; UND, unilaterally nephrectomized animals was less than their pretransplantation rate (p diabetic; UNC, unilaterally nephrectomized nondiabetic; N.S., not statistically significant, i.e., p < 0.05. < 0.05). Two months after transplantation the mean
i
SEPTEMBER, 1978
961
ALBUMINURIA IN DIABETIC RATS
of UND and UND-T animals were similar. Over the 12 Mean t SEM next two months, UAE rose markedly in UND animals compared with their seven-month values (figures 1 and 3 and table 1). In contrast (figure 3), UAE rate in UND-T rats fell within two weeks of trasplantation 10 compared with their pretransplant values (p < 0.05; UNILATERAL NEPHRECTOMY O) AND DIABETES paired /-test). Two months after transplantation UND-T rats had UAE rates that did not differ significantly from UNC animals but that were less than one-seventh those of UND rats (p < 0.02). The index of mesangial thickening (IMT) after E ra seven months of diabetes was 1.7 ± 0.1 in D rats as compared with 2.6 ± 0.01 in UND rats (p < < 7TRANSPLANTED 0.0005). Both D and UND animals had IMTs greater UNILATERAL NEPHRECTOMY AND DIABETES than their respective nondiabetic controls at this time (p < 0.0005 for each comparison). Two months after transplantation IMTs were 0.7 ± 0.2 in D-T and 1.1 ± 0.2 in UND-T animals, both being lower than their corresponding pretransplant values (p < 0.0005 and < 0.005, respectively). However, at this time UNILATERAL IMTs in D-T and UND-T rats remained higher than NEPHRECTOMY their corresponding nondiabetic littermates (p < 4 6 8 10 o 0.005 and < 0.0005, respectively). In the sevenMonths of Diabetes month compared with the nine-month time period FIG. 3. Urinary albumin excretion in unilaterally nephrectomized the IMT in D animals increased slightly to 2.0 ± nondiabetic, nontransplanted uninephrectomized diabetic, and uninephrectomized transplanted diabetic rats. At 0.08 (p < 0.05) while in UND rats no change in IMT transplantation (seven months) the uninephrectomized occurred. At the nine-month time period (two months transplanted animals were separated from the nontransplanted uninephrectomized diabetic group, and both groups were studied separately at that time and thereafter.
Mean i SEM
posttransplant) IMTs were lower in D-T and UND-T as compared with D and UND rats, respectively (p < 0.0005 for each comparison).
08
O.6
TRANSPLANTED DIABETES^
DISCUSSION r
c E
Eg 04
DIABETES
3 Q *
Q2
CONTROL
11
5 4
6
8
TO
Months of Diabetes FIG. 2.
962
Urinary albumin excretion in control, nontransplanted diabetic, and transplanted diabetic rats. At transplantation (seven months) the transplanted diabetic animals were separated from the nontransplanted diabetic group, and both groups were studied separately at that time and thereafter. Note change in the scale of the ordinate when compared with figures 1 and 3.
These studies confirm that proteinuria occurs in rats with experimental diabetes mellitus. In D animals UAE, higher than C throughout the study, increased further after seven months of diabetes. In contrast both UND and UNC rats had rising UAE rates with time, and the magnitude of the differences between these two groups increased progressively. Reversal of the diabetic state by pancreatic islet transplantation caused a return of UAE in both D-T and UND-T animals to their respective control levels within two months. Although paralleling the return to normoglycemia, the decline in UAE of D-T and UND-T rats lagged about two to three weeks behind the decline in plasma glucose values induced by transplantation. It is known that the major light, immunofluorescent, and electron microscopic alterations of diabetic DIABETES, VOL. 27, NO. 9
S. MICHAEL MAUER, M.D., A N D ASSOCIATES
glomerulopathy in rats are secondary to the diabetic state. Progressive mesangial thickening and mesangial deposition of immunoglobulins and complement 21 can be reversed after transplantation of a kidney from a diabetic to a normal host 27 or after successful pancreatic islet transplantation. 25 - 26 Further, GBM thickening in diabetic rats can be retarded in its development by instituting measures to lower blood sugar, including insulin administration and reduction in carbohydrate intake. 3 2 ' 3 3 Alterations in glomerular function in the earlier stages of diabetic nephropathy in man have been documented. 15 Studies of graded dextran polymer clearances in newly diagnosed or longer term diabetic patients have not indicated alterations in glomerular capillary filtration properties. 1 5 ' 3 4 The increased glomerular filtration rate, filtration fraction, and UAE with exercise in early diabetes have been interpreted as resulting from increased glomerular-filtering area and glomerular capillary pressures rather than from structural abnormalities of the glomerular capillary filter system. Azar* has documented elevated glomerular capillary pressures in diabetic rats. We have shown that unilateral nephrectomy accelerates the development of diabetic nephropathy in rats, 24 perhaps on the basis of increased glomerular blood flow and capillary pressures. 34 Thus it is possible that the exercise-induced albuminuria in diabetic man and the increased UAE in diabetic rats result from functional glomerular alterations and that unilateral nephrectomy, by accentuating these alterations, results in even greater urinary albumin loss. Although indirect evidence suggests that the mechanism for tubular reabsorption of protein in rats normally is not operating at a maximum, 36 ' 37 direct Bowman's capsule or proximal tubule micropuncture studies have shown that even minimally increased glomerular permeability to albumin results in increased quantities of albumin in the final urine. 3 8 ' 3 9 It is possible that exercise is required to unmask abnormalities in transglomerular capillary passage of albumin in man with well controlled diabetes 13 because a tubular threshold for albumin reabsorption must be surpassed. Diabetic patients in poor metabolic control, a situation more analogous to the present studies in rats, do have increased albuminuria at rest, apparently due to increased quantities of albumin in glomerular filtrate and, to a lesser extent, decreased tubular reabsorptive capacity for proteins. 15 ' 40 This albuminuria, associated with increased kidney size, *Azar, S., personal communication with me (SMM). SEPTEMBER, 1978
glomerular filtration rate, and filtration fraction, is, at least in part, reversible by the establishment of good metabolic control. 15 ' 40 Thus, the reversal of increased UAE by the achievement of normoglycemia after islet transplantation may be due to reversal of functional changes in glomerular hemodynamics. However, improvement in certain aspects of the glomerular pathology of diabetes in the rat has been documented to occur as early as two weeks after pancreas transplantation 26 and was clearly evident two months after transplantation in the present experiments. Thus, we cannot exclude the possibility that structural changes in the glomerular capillary filter system contribute to increased albuminuria in diabetic rats and that reversal of these changes by pancreas transplantation eliminates the increase. At this time it is not clear whether the increased UAE rate noted in our diabetic animals is analagous to albuminuria in the early stages of diabetic nephropathy in man or whether this increase more closely resembles the proteinuria that presages early renal failure in humans with severe diabetic glomerulopathy. 1 ' 2 Studies of differential renal protein clearances in diabetic patients have demonstrated differences in urinary IgG-transferrin clearance ratios in the early as compared with the late stages of diabetic nephropathy. 41 Studies along these and other lines may help to further elucidate the phenomena reported herein. ACKNOWLEDGMENTS We thank Ms. C. Swope, S. Sandberg, M. Stahlman, M. Rudquist, and Mr. J. Basgen for their excellent technical assistance. The rabbit anti-rat albumin antibody was generously supplied by Dr. J. R. Hoyer, Boston. We appreciate the secretarial and editorial assistance of Ms. Trudy Bensfield. Susan Perry drew the figures. We thank Esther Freier and Lorraine Gonyea-Stewart for their help with the statistical validation of the urinary albumin methodology. This work was supported by N . I . H . grants AM 17697 and HL-06314. REFERENCES ^ u s s m a n , M. J., Goldstein, H. H . , and Gleason, R. E.: The clinical course of diabetic nephropathy. JAMA 236:1861-63, 1976. 2 Knowles, H. C , Jr.: The magnitude of the renal failure problem in diabetic patients. Kidney Int. 6 (Suppl. l):2-7. 1974. 3 Rogers, J., Robbins, S. L., and Jeghers, H.G Intercapillary glomerulosclerosis: a clinical and pathologic study. II. A clinical study of 100 anatomically proven cases. Am. J. Med. 72:692-99, 1952. 963
ALBUMINURIA IN DIABETIC RATS 4
Bell, E. T.: Renal vascular .disease in diabetes mellitus. Diabetes 2:376-89, 1953. 5 Taft, H. P., Finckh, E. S., and Joske, R. A.: A biopsy study of the kidney in diabetes mellitus. Australas. Ann. Med. 3:189201, 1954. 6 Lambie, A. T., and MacFarlane, A.: A clinico-pathologic study of diabetic glomerulosclerosis. Q. J. Med. 24:125-42, 1955. 7 Gellman, D, D . , Pirani, C. L., Soothill, J. F., Muehrcke, R. C , and Kark, R. M.: Diabetic nephropathy: a clinical and pathological study based on renal biopsies. Medicine (Baltimore) 38:321-67, 1959. 8 Hennigar, G. F., Cohen, R. J., and Katz, H. P.: Nodular glomerulosclerosis: clinico-pathological correlation of 40 advanced cases. Am. J. Med. Sci. 42:89-99, 1961. 9 Thomsen, A. C : The Kidney in Diabetes Mellitus. Copenhagen, Munksgaard, 1965, pp. 208-15. 10 Yamauchi, Y., Suzuki, J., Ohneda, A., Yamagata, S., and Goto, Y.: Onset and progress of diabetic glomerulosclerosis. Toh o k u j . Exp. Med. 209:385-406, 1973. n Miles, D. W . , Mogensen, C. E., and Gunderson, H. J. G.: Radioimmunoassay for urinary albumin using a single antibody. Scand. J. Clin. Lab. Invest. 26:5-11, 1970. 12 Mogensen, C. E.: Urinary albumin excretion in early and long-term juvenile diabetes. Scand. J. Clin. Lab. Invest. 28:183-93, 1971. 13 Mogensen, C. E., and Vittinghus, E.: Urinary albumin excretion during exercise in juvenile diabetes: a provocative test for early abnormalities. Scand. J. Clin. Lab. Invest. 3.5:295-300, 1975. 14 0sterby, R.: Early phases in the development of diabetic glomerulopathy. Acta Med. Scand. Suppl. .574:1-80, 1975. 15 Mogensen, C. E.: Renal function changes in diabetes. Diabetes 25 (Suppl. 2):872-79, 1976. 16 Galaske, R. G., Gartner, K., and Stolte, H.: Functional and morphological study on the onset of proteinuria in experimental diabetes mellitus. In Contr. to Nephrology, Vol. 1. Eisenbach, G. M., Brod, J., and Stolte, H., Editors. Basel, Karger, 1975, pp. 69-79. 17 Weil, R., I l l , Nozawa, M., Koss, M . , Weber, C , Reemtsma, K., and Mclntosh, R.: The kidney in streptozotocin diabetic rats. Morphologic, ultrastructural and functional studies. Arch. Pathol. Lab. Med. 200:37-49, 1976. 18 Griswold, W. R., and Mclntosh, R. M.: Increased urinary excretion of a basement membrane-like glycoprotein in acute uranium nephropathy. Experentia 29:575-76, 1973. 19 Hagg, E.: Renal lesions in rats with long-term alloxan diabetes. Acta Pathol. Microbiol. Scand. 82:199-209, 1974. 20 Bloodworth, J. M. B . , J r . : Experimental diabetic glomerulosclerosis. II. The dog. Arch. Pathol. 79:113-25, 1965. 21 Mauer, S. M., Michael, A. F., Fish, A. J., and Brown, D. M.: Spontaneous immunoglobulin and complement deposition in glomeruli of diabetic rats. Lab. Invest. 27:488-94, 1972. 22 Mauer, S. M., Steffes, M. W . , Michael, A. F., and Brown, D. M.: Studies of diabetic nephropathy in animals and man. Diabetes 25 (Suppl. 2):850-57, 1976. 23 Scheinman, J. I., Steffes, M. W . , Brown, D. M., and Mauer, S. M.: The immunohistopathology of glomerular antigens. III. Increased mesangial actomyosin in experimental diabetes in the rat. Diabetes 27:632-37, 1978. "Steffes, M. W . , Brown, D. M., and Mauer, S. M.: Diabetic glomerulopathy following unilateral nephrectomy in the rat.
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Diabetes 27:35-41, 1978. 25 Mauer, S. M., Sutherland, D. E. R., Steffes, M. W . , Leonard, R. J., Najarian, J. S., Michael, A. F., and Brown, D. M.: Pancreatic islet transplantation: effects on the glomerular lesions of experimental diabetes in the rat. Diabetes 23:748-53, 1974. 26 Mauer, S. M., Steffes, M. W . , Sutherland, D. E. R., Najarian, J. S., Michael, A. F., and Brown, D. M.: Studies of the rate of regression of the glomerular lesions in diabetic rats treated with pancreatic islet transplantation. Diabetes 24:280-85, 1975. "Lee, C. S., Mauer, S. M., Brown, D. M., Sutherland, D. E. R., Michael, A. F., and Najarian, J. S.: Renal transplantation in diabetes mellitus in rats. J. Exp. Med. 239:793-800, 1974. 28 Matas, A. J., Payne, W. D., Grotting, J. C , Sutherland, D. E. R., Steffes, M. W . , Hertel, B. F., and Najarian, J. S.: Portal versus systemic transplantation of dispersed neonatal pancreas. Transplantation 24:333-37, 1977. 29 Mancini, G., Carbonara, A. O., and Heneman, J. R.: Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 2:235-54, 1965. 30 Hoyer, J. R., Mauer, S. M., and Michael, A. F.: Unilateral renal disease in the rat. I. Clinical, morphologic and glomerular mesangial functional features of the experimental model produced by renal perfusion with aminonucleoside. J. Lab. Clin. Med. 85:756-68, 1975. 31 ASTM Manual on Quality Control of Materials. Philadelphia, American Society for Testing Materials, I960, p. 63. 32 Hagg, E.: Influence of insulin treatment on glomerular changes in rats with long-term alloxan diabetes. Acta Pathol. Microbiol. Scand. 32:228-34, 1974. 33 Fox, C. J., Darby, S. C , Ireland, J. T., and Sorksen, P. D.: Blood glucose control and glomerular capillary basement membrane thickening in experimental diabetes. Br. Med. J. 2:605-07, 1977. 34 Mogensen, C. E.: Kidney function and glomerular permeability to macro-molecules in early juvenile diabetes. Scand. J. Clin. Lab. Invest. 28:79-90, 1971. 35 Azar, S., Johnson, M. A., Hertell, B., and Tobian, L.: Single-nephron pressures, flows and resistances in hypertension with nephrosclerosis. Kidney Int. 22:28-40, 1977. 36 Mendel, D.: Tubular reabsorption of protein in rats with experimental proteinuria. J. Physiol. (London) 2.56:44-54, 1961. 37 Bordeau, J. E., and Carone, F. A.: Protein handling by the renal tubule. Nephron 23:22-34, 1974. 38 Oken, D. E., Cotes, S. C , and Mende, C. W.: Micropuncture study of tubular transport of albumin in rats with aminonucleoside nephrosis. Kidney Int. 22:291-304, 1972. 39 Stolte, H., Kuhn, K., Galaske, R., Alt, J., Baldamus, C , Eisenbach, G. M., and Sterzel, R. B.: Function and structure following glomerular permeability changes in anti-GBM nephritis and aminonucleoside nephrosis. In Contr. to Nephrology, Vol. II. Sterzel, R. B., Thomson, D., and Brod, J., Editors. Basel, Karger, 1976, p. 114. 40 Parving, H. H . , Noer, I., Deckert, T., Evrin, P. E., Nielsen, S. L., Lynsg0e, J., Mogensen, C. E., R0rth, M., Svendsen, P. A., Trap-Jensen, J., and Lassen, N . A.: The effect of metabolic regulation on microvascular permeability to small and large molecules in short-term juvenile diabetes. Diabetologia 22:161-66, 1976. 41 Jerums, G., Post, R. S., Miller, M., and Barzellato, E.: Differential renal protein clearance in diabetes. Diabetes 22:10410, 1973. DIABETES, VOL. 2 7 , NO. 9
