THE ANATOMICAL RECORD 208:33-47 (1984) The Effect of Alloxan, and Alloxan-Induced Diabetes on the Kidney ANDREW P. EVAN, STEPHEN A. MONG, BRET A. CONNORS, GEORGE R. ARONOFF, AND FRIEDRICH C. LUFT Departments of Anatomy and Medicine, Indiana University School of Medicine, Indianapolis, IN 46223 ABSTRACT Alloxan is known to induce diabetic renal changes as well as causing nephrotoxic alterations. However, no ultrastructural study has been performed to differentiate diabetic verses toxic affects of alloxan to the tubule and/or glomerulus. Therefore the present study used the “protected” kidney model to prevent one kidney from being exposed to the alloxan while allowing the other to receive the drug immediately. In all experimental animals the right renal hilum was gently occluded for 5 minutes and then released. This was performed prior to the injection of alloxan. Subsequently, the left renal hilum was occluded at the time of, and for 5 minutes after, alloxan adminstration (40 m g k g i.v.). The experimental rats were divided into three groups: untreated diabetics, diabetics treated with protamine-zinc-insulin, and alloxantreated rats that failed to become diabetic. Three groups of controls were included: one group received a n equal volume of saline diluent as the experimental rats but no clamping of either renal hilum; another group received the saline and had the left renal hilum occluded for 5 minutes; and a third group had both the right and left renal hila occluded. All animals were followed and sacrificed after 9 weeks. Endogenous creatinine clearance did not change among groups. Alloxan-treated nondiabetic rats displayed marked interstitial nephritis in unprotected kidneys, while protected kidneys were normal. The diabetic state resulted in mesangial proliferation and focal glomerular basement membrane thickening as well as glomerular capillary endothelial abnormalities and visceral epithelial foot-process fusion. The endothelial changes consisted of focal areas showing a reduction in the size of endothelial fenestrae. All glomerular changes were ameliorated by insulin treatment. We conclude: 1)alloxan per se is distinctly nephrotoxic; and 2) the glomerular endothelium and epithelium are involved early in the course of experimental diabetes. Diabetes mellitus is responsible for wellrecognized renal functional and ultrastructural changes in man and experimental animals (Mogensen, 1976; Osterby, 1974). In animal models, diabetes is generally induced by the administration of agents that are toxic to pancreatic islet cells. Both alloxan and streptozotocin are useful for this purpose; however, both of these materials are also capable of causing renal damage apart from that created by the diabetic state (Rerup, 1970). Light microscopic studies by Orskov et al. (1965) and Vargas et al. (1970) have shown both tubular and interstitial changes 0 1984 ALAN R. LISS, INC. in the nonprotected kidney of a n alloxaninjected rat that would appear to be the result of direct alloxan damage to that kidney. In particular, Vargas et al. (1970) described cystic tubular dilations of the nephron leading to a spongy appearance of the cortex which was related to alloxan toxicity. More recent studies have employed both transmission and scanning electron microscopy to follow changes in the kidney during alloxan-induced diabetes (Hagg, 1974; Hagg Received May 24, 1983;accepted September 2,1983. 34 A.P. EVAN ET AL. and Winbland, 1975; Evan and Luft, 1980; Bell et al., 1980). However, no study has differentiated the fine structural changes that may result from alloxan toxicity versus diabetes. In the present experiments, we have extended our ultrastructual observations of alloxan diabetic rats to animals in which one kidney was protected by means of a postalloxan-injection, renal-hilar-clamping technique. METHODS General Male Sprague-Dawleyrats, initially weighing 200-225 gm, were used in the present study. For the induction of diabetes, the animals were first anesthetized with pentabarbital. All rats were allowed free access to tap water and a standard rat diet (Purina). Following assignment to their groups, the rats were placed in metabolic cages for 24-hour urine collections. Blood was also obtained at this time from the tail vein. These collections were repeated at weeks 1 through 8, following which the rats were sacrificed for morphological studies. No more than three deaths occurred prior to sacrifice in any group. Only animals which were studied at every time point were included in the data analysis. Alloxan- Treated Animals diabetic. The last group was also diabetic, but these animals were given protamine-zinc insulin (Eli Lilly & Company) as a daily subcutaneous injection (1-5 units). The amount of the insulin dose was adjusted for each animal so that their plasma and urine glucose concentrations were maintained at control levels. Control Animals Three groups of control animals were used with 20 rats per division. The first group of rats were age-matched controls that received 1ml of 0.9% saline solution by tail-vein injection but did not have either renal hilum clamped. Another group had only the left renal hilum occluded for 5 minutes in addition to receiving 1 ml of the saline solution. The third group had the right renal hilum occluded for 5 minutes followed by clamping of the left hilum for 5 minutes while receiving 1ml of the saline solution. An occlusion time of 5 minutes was chosen for several reasons. First, it has been shown that the concentration of alloxan rises rapidly in the plasma, kidney, and pancreatic islets during the first 5 minutes after a bolus injection. The concentrations in the plasma and kidney then falls quickly over the next 2 hours but not in the pancreas (Janes and Winnick, 1952; Hammarstrom and Ullberg, 1966; Bilic and Felber, 1969). Therefore the 5-minute interval would prevent the clamped kidney from accumulating the drug while allowing the other kidney t o excrete the compound. The clamped-protected kidney would be exposed to alloxan after the time of clamp removal; however, no toxicity to that organ was observed, according to our morphological results. Second, alloxan causes most of its tissue necrosis, particularly in the pancreas, within 5 minutes of injection (Hughes et al., 1944). Third, Orskov et al., (1965) and we (unpublished observations) have noted that if the occlusion time was kept to 5 minutes or less, there was minimal postoperative mortality and a high percentage of animals developed insulin-dependent diabetes. Last, several authors have previously shown that a 5-minute or shorter occlusion time was sufficient to protect the kidney from alloxaninduced nephrotoxicity (Orskov et al., 1965; Arteta, 1952). After anesthesia, both renal hila were exposed via a midline abdominal incision. First, the right renal hilum was gently occluded for 5 minutes via a spring-clamp. The occlusion was released and then applied t o the left renal hilum as alloxan monohydrate (Sigma) at 40 mgkg in 0.9% saline solution was rapidly injected as a bolus into the vena cava. After 5 minutes the clamp was removed and the incision closed in two layers. The 40-mgl kg dose and bolus injection protocol was used because of its effectiveness in inducing diabetes in the rat compared to giving several smaller doses over time which creates a subdiabetic state. Eighty animals received alloxan, and 4 days later the presence of diabetes was verified by determining the nonfasting serum glucose level. Only those rats with a glucose concentration of at least 250 mgldl were considered diabetic. At this time, three separate groups were formed. The Fixation Procedures first group contained animals that had reAll kidneys were preserved by in vivo perceived alloxan but developed neither glycosuria nor hyperglycemia and therefore were fusion. Following pentabarbital anesthesia, termed nondiabetic alloxan-treated. The sec- the abdominal cavity was opened and a polyond group was made up of animals that were ethylene catheter was introduced into the ALLOXAN-INDUCED DIABETES abdominal aorta below the renal arteries. Using 100 mm Hg pressure, 30 ml of 0.9% NaCl was perfused through the catheter followed by 150 ml 2.5% glutaraldehyde in 0.075 M cacodylateEIC1 buffer, pH 7.4. Following fixation, portions of the outer and inner cortex were removed and further fixed in the original fixative for a n additional 48 hours. At this time, all specimens were assigned coded numbers and examined without knowledge of the regimens. One-millimeter cubes of tissue were routinely prepared for transmission electron microscopy and viewed with a Philips EM-400 electron microscope. Larger pieces were washed in the buffer for 90 minutes, dehydrated through a series of graded alcohols to 100%ethanol, fractured in liquid nitrogen, transferred to a Samdri-critical point dryer and dried with liquid COz. Tissues were attached to a n aluminum stub and placed in a sputter coater (Hummer V) and coated with gold-palladium. Specimens were examined on a n AMR-1000A scanning electron microscope. Sections were also obtained for light microscopy and were stained by hematoxylin and eosin or by the periodic acid-Schiff (PAS) reaction. 35 In addition to the semiquantitative assessments, we also performed quantitative measurements to determine basement-membrane thickness. This determination was accomplished using a computer digitizing system and Quantigraph program (Novus Instruments, Carmel, IN). Thickness of the glomerular basement membrane was determined from transmission electron micrographs printed at a fixed magnification of x 30,000. Approximately ten micrographs were obtained from each of five randomly selected glomeruli per kidney. Ten measurements were made along a 10-mm length of normal or thickened glomerular basement membrane. Only those lengths of basement membrane were used which appeared perpendicular to the capillary wall. All measurements were obtained on a line orthogonal to the edge of the glomerular basement membrane starting at the endothelial surface. The line that was measured extended from the endothelial to the epithelial side. Chemical Analysis At weekly intervals, animals were placed in metabolic cages in order to obtain a 24hour urine collection. A blood sample was Tissue Analysis also obtained from the tail vein. Both the In order to obtain a statistical comparison blood and urinary glucose levels were deterof some of the changes noted in the kidneys mined on a Beckman Glucose I1 analyzer of the different experimental groups as seen (Beckman Instruments, Inc., Creve Coeur, by light, transmission, and scanning electron MO). The creatinine levels were measured microscopy, we employed a semiquantitative with a Beckman Creatinine I1 analyzer. The histologic analysis (Bohman et al., 1979; Pir- data were analyzed by means of repeated ani et al., 1964). The severity and distribu- analysis of variance and Student’s t-test as tion of each specific lesion was scored by the indicated. The 95% limits of probability were following scale: 0 = absence of the lesion, 1 accepted as significant. = lesion represented in less than 10% of the nephrons and with minimal change, 2 = leRESULTS sion represented in up to 50% of the nephThe plasma glucose concentrations appear ions and with moderate change, 3 = lesion represented in 50 to 90% of the nephrons in Figure 1. Untreated diabetes resulted in with moderate change, and 4 = lesion repre- marked hyperglycemia throughout the pesented in over 50%of the nephrons and with riod of observation. The range of plasma glusevere change. The lesions observed by light cose levels during the last week was 280-628 microscopy included tubular atrophy, tubu- mg/dl. Insulin treatment brought blood sugar lar dilation, interstitial nephritis, glomeru- values into the normal range except during lar sclerosis, and distal tubular vacuolation. week 4. The control group in which both Scanning and transmission electron micros- renal hila were clamped had values of 90copy were used to examine the visceral epi- 150 mg/dl throughout the experiment. Those thelium to detect podocyte loss or fusion, the animals that received alloxan but had glomerular endothelium for alterations of the plasma glucose concentrations similar to that fenestral diameter and mesangial cells for a of the controls, formed a separate group, the change in number. A mean diameter of indi- nondiabetic alloxan-treated animals. The vidual endothelial fenestra was calculated age-matched controls, and controls with right after determining both a maximum and min- hilar clamping only, had similar values as imum caliper diameter off of micrographs those control animals in which both renal hila were occluded. printed a t a final magnification of 30,000. 36 A.P. EVAN ET AL. 500 -. 400--- 300- - -I 0 \ -E" - u g 200- -> CI 0 0 - E h 100 -. 0 1 2 3 4 5 6 7 6 Weeks 0 Diabetes Untreated 0 Diabetes Treated A Alloxan No Diabetes 0 Control w/Both Renal Hila Clamped - Fig. 1. Plasma glucose concentrations of diabetic, nondiabetic alloxan-treated, insulin-treated diabetic, and control rats. Table 1outlines the effects on body weight, urine volume, creatinine clearance and glucose excretion. Only data from week 0 and the week of sacrifice are shown. Increases in body weight were observed in every group (P < .001); however, the increase was least in the diabetes untreated group (P < .05). The diabetic state resulted in polyuria, which was ameliorated by insulin treatment (P < .001). No significant interactions between treatment and creatinine clearance were observed. Urine glucose excretion was in the range of 7,500 mglday in untreated diabetic animals. Treatment brought glycosuria to within the range observed in all control rats. Figures 2-7 are light photomicrographic sections from the protected and nonprotected kidneys of nondiabetic animals killed 8 weeks following alloxan treatment. The entire protected kidney appears normal (Figs. 2, 4) while the unprotected kidney shows areas of normal tubules adjacent to extensively injured tubules (Figs. 3, 5-7; Tables 2, 3). Damage to the nephron was noted by tubular atrophy, dilation, and glomerular sclerosis (Figs. 3, 5-7). A few tubules were filled 37 ALLOXAN-INDUCED DIABETES TABLE 1. Changes in body weight, urine volume, creatinine clearance, and glucose excretion at week 0 compared to week 8 in all experimental and control groups (mean f SEM) Group Diabetes Untreated Insulin-treated Nondiahetic Alloxan-treated Control, Both renal hila clamped Left hilum clamped Age-matched (n) Week 0 Variable Weight (gm) Urine volume (m1124 hr) Creatinine clearance (ml/min) Urine glucose (mg/24 hr) (17) Weight (gm) Urine volume (m1/24 hr) Creatinine clearance (mlimin) Urine glucose (mg/24 hr) (13) Weight (gm) Urine volume (mli24 hr) Creatinine clearance (mlimin) Urine glucose (mgi24 hr) (20) Weight (gm) Urine volume (m1124 hr) Creatinine clearance (mlimin) Urine glucose (mgi24 hr) (20) Weight (gm) Urine volume (m1124 hr) Creatinine clearance (mlimin) Urine glucose (mg/24 hr) (10) Weight (gm) Urine volume (mli24 hr) Creatinine clearance (mlimin) Urine glucose (mgi22 hr) with cast material (Fig. 5). An interstitial nephritis characterized by a cellular infiltration (mainly lymphocytes) and fibrosis was associated with the tubular injury and extends from the cortex to the medulla of the kidney. Figure 7 shows the same area by both light microscopy and transmission electron microscopy of an unprotected kidney obtained from an animal that received alloxan but did not develop diabetes. A proximal tubule recognized by apical microvilli shows considerable evidence of atrophy, noted by its reduced size. The individual cells present an Week 8 222 I 8 308 f 22 110 f 12 128 + 8 0.535 f 8,720 218 103 0.10 f 1,340 7,495 8 12 421 22 f i 0.554 f 0.2 f 1,600 8,800 0.481 t 0.04 t 1,160 * 0.577 18 3 0.09 86 f 29 230 k 9 472 f 15 10 f 1 13 f 2 0.547 0.12 0.513 f 0.05 74 I 17 68 k 9 217 5 3 498 i 14 t 5 12 f 1 7 - 0.528 f 0.10 0.581 t + 9 82 k f 3 423 f f 1 9 i 1.67 f 0.2 107 202 7 2.79 5 0.06 20 6 1 0.3 8 f 1 12 f 1 208 9 f 4 5 430 6 I 8 1 i 1.58 f 0.3 6 1 + 5 2.01 t 10 f 0.3 2 apparent reduction in the number of organelles and interdigitating processes. The interstitium contains numerous cells not normally found there as well as an increased amount of collagen material. Scanning electron microscopy reveals a loss of microvilli in approximately 10% of the injured proximal tubules from the unprotected kidney when compared t o the protected of the same nondiabetic animal (Figs. 8,9). The renal corpuscles from the protected kidney are normal by both transmission and scanning electron microscopy except for pericapsular fibrosis. The Fig. 2. Light micrograph showing the cortex and medulla of the left (protected) kidney from an animal that did not develop diabetes, sacrificed 8 weeks after receiving alloxan. No glomerular (G) or tubular alterations were noted. x 10. Fig. 3. Light micrograph showing a similar region of the right kidney from the same animal a s in Figure 2. Areas of tubular atrophy and interstitial nephritis extend from the cortex to the outer medulla (arrows). Some tubules are dilated (D), and contain cast material within their lumens. x 10. Fig. 4. Higher-magnification light micrograph froin the same kidney as in Figure 2. No obvious changes are noted in the renal corpuscle, tubular segments, or interstitium. x25. Fig. 5. Higher-magnification light micrograph from the same kidney as in Figure 3. Normal proximal tubules (P)are seen adjacent to atrophic tubular segments (arrow). A tubule filled with cast material is seen. ~ 2 5 . Fig. 6. A plastic thick section from the same kidney as in Figure 3. Normal proximal tubules are seen next to atrophic tubular segments (arrow). The injured tubules are surrounded by an interstitial nephritis which is characterized by a cellular infiltrate and fibrosis. Some of the injured proximal tubules show almost a complete loss of their brush border (double arrow). x80. 40 A.P. EVAN ET AL. Fig. 7. A light microscopic and transmission electron micrograph (TEM) from an unprotected (right) kidney of an animal that did not develop diabetes, sacrificed 8 weeks after receiving alloxan. A low-magnification light microscopic inset shows the same tubules seen in the TEM. Two atrophic segments are seen near a cortical collecting tubule (CT). One of the injured tubules can be identified as a proximal tubule (double arrow) due to the presence of apical microvilli. This tubule is considerably smaller than the more normal appearing proximal tubules (PI. The other atrophic segment (arrow) is difficult to classify. TEM, X 3,800; LM, ~ 7 5 . 41 ALLOXAN-INDUCED DIABETES TABLE 2. Semiquantitative changes in renal architecture: Mean pathological score Control Chance Tubular atrophy Tubular dilation Interstitial neDhritis Glomerular sclerosis Distal tubular vacuolation Foot process loss Fenestral diameter reduction Mesangial cell proliferation Nondiabetic Diabetic Treated diabetic Rt Lf Rt Lf Rt Lf Rt Lf 0.00 0.00 0.00 0.00 3.25 o.oo* O.OO* 3.50 2.50 0.00 2.00 o.oo* 3.00 2.00 0.00'; 0.00 0.00 3.75 3.50 0.00' 3.50 o.oo* 0.00 0.00 3.00 o.oo* o.oo* 3.25 0.50* 3.00 0.25* 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3.25 2.25 3.00 2.00 1.00 0.00 0.50 0.00 0.00 0.00 0.00 0.00 1.25 1.50 0.00 0.25 0.00 0.00 0.00 0.00 2.50 2.00 0.00 0.00 o.oo* "N represents the number of animals used. The control group includes all three control groups: age-matched, right hilum clamp Ynly, both right and left hila clamp. Significant difference right vs. left or left vs. right (P< .05). Five sections from each kidney were examined. TABLE 3. Basement membrane thickness (BMT) for right (Rt) and left (LD kidneys BTM (nm) Group Controlb Nondiabetic Diabetic Treated diabetic Rt Lt Na 193 2 3' 195 k 5 190 i 5 235 k 3* 195 4 10 188 k 4 241 + 6* 200 +7 + 10 10 10 sN represents the number of animals used. bIncludes only those animals in which both renal hila were clamped. 'Values expressed as the mean f SEM. "P < .05 for difference between control and diabetic animals. mesangial cells, filtration barrier, and glomerular vasculature exhibit no changes. The protected kidneys from the alloxan nondiabetic group resembles that of the control group (Tables 2, 3; Figs. 2, 4).The data from all three control groups were combined in that no structural changes were found in any of the kidneys. The 5-minute clamping period did not appear to induce chronic injury to the tubules or glomeruli. Diabetic untreated animals also exhibited severe interstitial nephritis in the unprotected kidney after 8 weeks (Table 2). However, in protected kidneys, interstitial nephritis was not a feature. The cytoplasm of tubular epithelium from some distal tubules and collecting ducts from the protected kidney exhibited large clear vacuoles (PAS-negative) by light microscopy which by transmission electron microscopy had a flocculent appearance. These electron microscopic changes are shown in Figure 10. In addition the protected kidney from the untreated diabetic animals possessed glomeruli that exhibited a n increased number of mesangial cells and focal areas of glomerular basement membrane thickening and podocyte fusion (Fig. 11). The four panels of Figure 12 display the glomerular endothelial surface from control and experimental groups as viewed by scanning electron microscopy. Panel A is from a control animal in which both the right and left hila were clamped. The en$othelial fenestrae are approximately 750 A in diameter. Occasional cytoplasmic ridges are observed. Panel B shows the glomerular endothelium from the protected kidney of a n animal after eight weeks of untreated diabetes. There are areas of the capillary where the endothelial fenestrae sbow a reduction in size to as small as 350 A in diameter. These changes are focal in that a capillary loop may possess both normal and abnormal cells (Table 3). The semiquantitative data reveals the relative increase or absence of injured endothelial cells. Small spherical structures, which appear to be platelets, can be seen attached to the luminal surface of the endothelial cells. Further injury of the endothelium is seen in panel C, where denuded areas are found along the capillary wall. These cells are from the same animal illustrated in panel B. Panel D is from a n insulin-treated animal, and all endothelial cells resemble those of the control animals (Table 3). Figure 13 shows the visceral epithelial surface of a n untreated diabetic animal. Panel A shows a control animal and reveals numerous slender interdigitating foot processes. However, the untreated diabetic animals possess focal areas of clearly blunted and 42 A.P. EVAN ET AL. Fig. 8. Scanning electron micrograph of a proximal tubule from a protected (left) kidney of an animal that did not develop diabetes, 8 weeks after receiving alloxan. The apical surface presents an elaborate arrangement of microvilli. ~ 3 , 0 0 0 . Fig. 9. A proximal tubule from the unprotected (right) kidney from the same animal as in Figure 8. These cells show an extensive loss of microvilli (arrow). X3,OOO. fused foot processes (B). Such changes are not observed in animals of the nondiabetic group or in those which received insulin treatment (Table 3). Tables 2 and 3 summarize by both semiquantitative and quantitative analysis the changes in renal morphology just described. By light microscopy it was noted that only the right, or unprotected, kidney from all animals receiving alloxan showed changes consisting of tubular atrophy and dilation, interstitial nephritis and glomerular sclerosis. These data plus the fact that the left, or protected, kidney from the nondiaetic rats showed no alteration clearly points out the nephrotoxic effects that alloxan has by itself on the kidney. The diabetic changes are obvious when the protected kidney from the nondiabetic animal is compared to that of the diabetic rat. The protected kidney from the diabetic animals showed glomerular changes which in- cluded a loss of the epithelial foot processes, reduction in the diameter of the endothelial fenestrae (Table 21, increase in basement membrane thickness and proliferation of mesangial cells (Table 3), as well as vacuolation of the distal portion of the nephron. Fig. 10. Cortical collecting tubule from the protected kidney of an untreated diabetic animal sacrificed 8 weeks after the alloxan injection. Most cells of this tubule are abnormal in that they display a lucent cytoplasm with many organelles (arrows) positioned at the cell periphery. ~ 2 , 0 0 0 . Fig. 11. Transmission electron micrograph of a portion of a glomerulus from the same kidney as in Figure 10. Two capillary loops are seen in cross section. The basement membrane of the capillary on the right is generally uniform in width while the one on the left shows several areas (arrows) of increased thickness. The foot processes (P) of both loops are irregular in width. x 12,000. Fig. 12. A) Scanning electron micrograph of glomerular endothelium from the left kidney of a control animal in which both renal hila were clamped for 5 minutes; the animal was sacrificed 8 weeks later. The cells display numerous large fenestrae (arrow) and occasional cytoplasmic ridges (C). x 18,000. B) Glomerular capillary from the protected kidney of an untreated diabetic rat sacrificed 8 weeks after receiving alloxan. There is a reduction in size of the fenestrae (arrow). Spherical structures resembling platelets are seen (double arrow). x 18,000. C) Endothelium from the protected kidney of an untreated diabetic rat (same as animal in Fig. 12B) showing focal degeneration (arrow) and exposure of the underlying basement membrane. X 18,000. D) Glomerular capillary from the protected kidney of a n insulintreated diabetic animal sacrificed 8 weeks after receiving alloxan. The endothelium is normal in appearance. x 18,000. ALLOXAN-INDUCED DIABETES 45 Fig. 13. A) Visceral epithelium from the right kidney of a control animal (same as animal in Fig. 12A) in which both renal hila were clamped. The cells exhibit long, . Visslender, interdigitating foot processes. ~ 5 , 0 0 0 B) ceral epithelium from the protected kidney of an untreated diabetic animal (same as Fig. 12B). There are focal areas of blunting and fusion of the foot processes (arrow). ~ 5 , 0 0 0 . These changes are seen in both the right and left kidneys of the diabetic animals. Further evidence that these alterations are related to the diabetes is that the changes are reversible with insulin treatment (Tables 2, 3). Glomerular volume measured in all groups remained unchanged from the control values (Table 3). nephropathy, identified a number of potentially important glomerular endothelial and epithelial abnormalities which have not been previously appreciated. The experiments of Orskov et al. (1965)raised the possibility that the alterations we identified were related to alloxan rather than diabetes. These investigators used the approach to protect the kidney employed in the present study and observed tubular atrophy, dilation, and interstitial inflammation, which they attributed to alloxan. Current sophisticated ultrastructural techniques were not used by Orskov et al. (19651, and the possibility remained that significant glomerular damage occurred related to alloxan that would not be detected by light microscopy. Such has been the case in the study of other nephrotoxins, including the aminoglycoside antibiotics and heavy metals (Luft and Evan, 1980; Avasthi et al. 1980). The distinction between the effects of alloxan and diabetes per se is important, not only with respect to interpreting morphol- DISCUSSION Chemically induced diabetic models have commonly been employed to study the functional and structural changes associated with diabetes mellitus as well as the effects of treatment with either insulin or pancreatic islet cell transplantation (Black et al., 1980; Rasch, 1979; Mauer et al., 1974; Orloff et al., 1975; Weil et al., 1975). Recently we examined the renal glomerulus in a series of sequential studies which utilized rats made diabetic with alloxan (Evan and Luft, 1980). The scanning electron microscope, a tool not previously employed in the study of diabetic 46 A.P. EVAN ET AL ogy, but also in evaluating the results of renal functional studies including micropuncture experiments (Michaels et al., 1981). Our studies indicate that alloxan caused considerable interstitial nephritis and tubular atrophy. These changes were observed not only in rats that developed diabetes, but also in rats which received alloxan but failed to develop diabetes. Thus this latter group was particularly useful in defining the effects of alloxan. In those areas that showed severe interstitial changes, glomerular sclerosis was also prominent. However, these glomeruli had not presented the same changes that were associated with diabetes, such as podocyte fusion, mesangial hyperplasia, and basement membrane thickening. Therefore the nephrotoxic and diabetic changes could be differentiated. The diabetic state resulted in mesangial hyperplasia and focal glomerular basementmembrane thickening after only 9 weeks of untreated diabetes. These structural changes have been implicated in the functional changes observed in diabetes (Steffes et al., 1975). Experiments in which glucose and insulin concentrations in diabetic rats were controlled have shown decreases in mesangial matrix, immunoglobulin deposition and glomerular cellularity as well as improvment in renal function (Mauer et al. 1974; Orloff, 1975; Weil et al. 1975). In addition, a recent study examining the effects of insulin treatment demonstrated that good blood glucose control in rats perserved normal glomerular basement-membrane thickness (Rasch, 1979). The glomerular capillary endothelial changes, namely, a decrease in size of the fenestrae, proliferation of microvilli, and appearance of irregular endothelial cell processes, can now be attributed solely to the diabetic state. The endothelial structural aberrations may be partially responsible for abnormalities in glomerular function described in recent micropuncture studies (Hostetter et al., 1981). The pathogenesis of the endothelial changes is unknown. Similarly, little is known of the mechanisms responsible for the increase in amount of glomerular basementmembrane material. The latter has been attributed to abnormal deposition due to excessive synthesis of glomerular basementmembrane material as well as faulty disposal (Romen, 1980).The functional relationships between capillary endothelium and glomerular basement membrane are imper- fectly defined. Our results suggest that endothelial damage antedates glomerular basement-membrane thickening. Whether or not endothelial damage and altered glomerular basement-membrane metabolism are separate events or are specifically related is not known. Elucidation of a possible connection must await detailed studies of endothelial cell growth and metabolism. Streptozotocin is another commonly used drug to induce diabetes mellitus in laboratory animals. Several studies have shown this compound to produce direct injury to kidney, in particular, the proximal tubule (Sadoff, 1970; Myerowitz et al., 1975; Levine et al., 1980). Alternatively, Steffes et al. (1980) and Rasch (1979) have presented indirect data which suggests that streptozotocin may not have direct toxic effects on the kidney since the glomerular changes can be either ameliorated or prevented by treatment by insulin or islet transplantation. However, a protection study as performed in our present report has not been done for streptozotocin. In summary, our experiments differentiate the effects on renal tissue caused by alloxan from those evoked by diabetes. Alloxan induces marked interstitial nephritis but does not appear to affect glomerular structure. Untreated diabetes caused hyperplasia of mesangium, changes in glomerular basement membrane, visceral epithelial foot process fusion, and endothelial damage. These features were ameliorated by treatment with insulin. ACKNOWLEDGMENTS This study was supported by Diabetes Grant PHS P60 AM 20542-03. The authors wish to give special thanks to Ms. Toni D. Moore and Mrs. Sandra S. Wilson for typing the manuscript. LITERATURE CITED Arteta, J.L. (1952) Mechanism of protective action of clamping renal pedicles of dogs with alloxan diabetes. J. Endocrinol., 8.245-249. Avasthi, P.S., A.P. Evan, and D. Hay (1980) Glomerular endothelial cells in uranyl nitrate-induced acute renal failure in rats. J. Clin. Invest., 65:121-127. Bell, R.H., L. Fernandez-Cruz,-J.E.Brimm, H.A. Sayers, S. Lee, and M.J. Orloff (1980) Prevention of whole pancreas transplantation of glomerular basement membrane thickening on alloxan diabetes. Surgery, 88:31-40. Bilic, N., and J.P. 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