THE ANATOMICAL RECORD 214:341-347(1986) Morphology of Norepinephrine-lnduced Acute Renal Failure in the Dog RUTH ELLEN BULGER, THOMAS J. BURKE, ROBERT E. CRONIN, ROBERT W. SCHRIER, AND DENNIS C. DOBYAN Renal Research Laboratory, The University of Texas Health Science Center, Graduate School of Biomedical Sciences at Houston, Houston, T X 77030 (R.E.B., D.C.D.), The Department of Medicine, University of Colorado Health Sciences Center, Denver. CO 80220 (TJ.B., R. W.S.), and The Department of Internal Medicine, Veteran’s Administration Medical Center at Dallas, Dallas, TX 75216 (R.E.C.) ABSTRACT The 40-minute infusion of norepinephrine (NE) into the renal artery of dogs produces a reversible ischemic model of acute renal failure. While the physiology of this model has been extensively studied, no complete description of the pathology exists. This study uses light microscopy and transmission electron microscopy to describe and quantitate the structural and ultrastructural changes which occur in the kidneys of dogs 1, 3, and 24 hours after the intrarenal infusion of 0.75 mgkg/minute of NE. One hour after a 40-minute NE infusion the majority of convoluted and straight proximal tubules showed apical blebs, loss of brush border, microvillar whorl formation, and mitochondria1 condensation and high-amplitude swelling with flocculent densities. Necrotic cells were occasionally seen at 1 hour. The injury was progressive after 3 hours and by 24 hours animals had either complete or partial patchy necrosis of all regions of the proximal tubule. The percentages of injured and necrotic proximal tubules in outer, mid-, and inner cortical regions are presented. We conclude that the extent and pattern of injury seen after NE infusion differs significantly from the renal artery clamping model of ischemia. The infusion of norepinephrine (NE) (0.75 pgkgiminUte) into the renal artery of dogs for 40 minutes produces a reversible model of acute renal failure. Although the pathophysiology of this model has been extensively studied (Cronin et al., 1978a,b; Schrier et al., 1978; Burke et al., 1980, 1982; Patak et al., 1979; de Torrente et al., 1978),a complete description of the pattern and progression of renal injury caused by this treatment has not been made. This report concentrates on the renal injury caused by norepinephrine infusion and compares it to the injury previously described in the renal pedicle clamp model of rat (Glaumann and Trump, 1975; Glaumann et al., 1975; Venkatachalam et al., 1978, 1981). MATERIALS AND METHODS Experiments were performed on seven mongrel dogs of both sexes weighing 22-32 kg. Food was withheld for 18 hours and water was allowed ad libitum. Anesthesia was induced with intravenous sodium pentobarbital (25 mgkg). The animals were intubated and ventilated with a Harvard respirator. Light anesthesia was maintained by the intermittent administration of additional small doses of pentobarbital. Catheters were placed in both ureters and renal veins, and the left renal artery was isolated for placement of a n electromagnetic flow probe (Carolina Medical Electronics, Inc., King, NC). During dissection, care was taken to prevent damage to the renal nerves. After control clearance determinations, an angiographic catheter, French 7-8 (Cordis Corporation, Miami, FL), was introduced into a femoral artery and 0 1986 ALAN R. LISS, INC manually guided into one renal artery. The catheter was positioned in the main renal artery while avoiding obstruction of blood flow. Streaming of the injected NE in the renal artery did not appear to occur since the fall in renal blood flow (RBF) to zero following the NE infusion was rapid and complete within approximately 10-30 seconds. For the %hour studies, the catheter was placed by using fluoroscopy and dye injection. During the procedure, arterial blood pressure was measured from a brachial artery catheter with a Statham transducer (Statham Instruments, Inc., Oxnard, CAI. Surgical fluid losses were replaced with isotonic saline. Since these animals were used for studying the pathophysiology of this model, physiological parameters were measured as described elsewhere (Burke et al., 1984). Clearance of inulin and para-aminohippuric acid were measured by standard techniques. PAH clearances were corrected for extraction. Total plasma protein was measured by the Technicon autoanalyzer method. Plasma and urine osmolality determinations were made by using an osmometer (Advanced Instruments, Needham Heights, MA). The experiments were begun after a 60-minute equilibration period and control clearance collections. Norepinephrine (0.75 pg/kg/minute) was infused into one renal artery for 40 minutes. At the conclusion of the infusion Received J u n e 25, 1985; accepted October 23, 1985. Address reprint requests to Dr. Ruth Ellen Bulger, Renal Research Laboratory, The University of Texas Medical School, Room 7.242 MSMB, P.O. Box 20708, Houston, TX 77225. 342 R.E. BULGER ET AL. Fig. 1. Light micrograph showing the kidney from a dog subjected to norepinephrine (NE) infusion, Note the patchy cortical necrosis (arrows). Hematoxylin and eosin, x 130. Fig. 2. Light micrograph showing the kidney from a dog subjected to NE infusion. Note the extensive necrosis involving all of the proximal tubules. Hematoxylin and esoin, x 110. period the catheter was withdrawn and the kidneys were subsequently fixed at 1, 3, and 24 hours after completion of the norepinephrine infusion. For the fixation process, loose ligature snares were placed above and below a short segment of aorta that included both renal arteries. One-half-strength Karnovsky’s fixative (1965)buffered with potassium phosphate . was perfused into this aortic segment through a 16gauge needle, with a monitored perfusion pressure maintained equal to or slightly greater than mean arterial pressure. When the solution was flowing well, the ligature snares were tightened, maximizing flow of fixative to the renal arteries. The renal veins were then cut to allow free flow of fixative out of the kidneys. A total of 300-400 ml of fixative was used in each animal. Pieces of kidney from each animal were embedded in paraffin according to routine histologic methods. Sections 5 pm thick were cut and stained with hematoxylin and eosin for light microscopic analysis. Tissue for transmission electron microscopy was minced and washed in 0.2 M phosphate buffer, pH 7.4, for 1hour. The tissue was then postfixed in 2% osmium tetroxide buffered in 0.1 M s-collidine (pH 7.2-7.4) for 1 hour a t room temperature, stained en bloc with uranyl acetate in veronal acetate buffer at pH 5.0, dehydrated in a graded series of alcohols, treated with propylene oxide, and embedded in epoxy resin. Ultrathin sections were cut on Sorvall MT-2 or LKB I11 ultramicrotomes, stained sequentially in 7.5% uranyl magnesium acetate and 0.15% lead citrate, and examined with a Siemens 102 or a Philips 200 transmission electron microscope. Semithin epoxy sections (0.5-1.0 pm thick) also were cut, stained with toluidine blue, and viewed with a light microscope. QUANTITATION OF STRUCTURAL ALTERATIONS Paraffin sections of kidney were used for the quantitative analysis of injury. In each kidney, five areas each from the outer cortex, the midcortex, and the inner cortex were utilized. The data from each region was then combined to give a single value for each. Tubular injury was evaluated in the proximal tubule, the major site of injury along the nephron. By using a Leitz-Wetzlar overhead projecting microscope, each area was first centered a t low-power magnification and then the high-power objective was positioned for viewing the field a t random, without moving the specimen stage to avoid bias of the chosen area. The image was projected upon a screen containing 144 points. The cell or structure lying under or nearest each point was classified by one observer (R.E.B.) without the observer having prior knowledge of the treatment of the specimen being examined. Each of the counted proximal tubular cells was assigned to one of the following categories: 1)normal or indistinguish- NOREPINEPHRINE-INDUCED ACUTE RENAL FAILURE IN DOGS able from controls, 2) injured when the cell shape was obviously altered to a low cuboidal or squamous type or revealed extensive apical vesiculation or vacuolization in addition to the loss of brush border but had no evidence of necrosis, or 3) necrosis when the cell showed irreversible damage such as loss of membrane integrity, or loss of nuclear staining, or had been shed into the lumen. All values reported represent mean & standard error of the mean. This method, a modification of the point-counting method described by Weibel (19791, has been used extensively in our laboratory to quantitate renal structural changes (Eknoyan et al., 1982; Dobyan and Bulger, 1984). RESULTS General comments Some glomeruli in both the infused and contralateral kidneys of several of the dogs exhibited diffuse hypercellularity. There were abundant neutrophilic polymorphonuclear leukocytes seen in both the control and experimental kidneys. The contralateral kidneys, however, failed to show any significant degree of acute cellular injury. The changes observed were similar regardless of the sex of the animal under investigation. In some animals, casts and calcified material could be seen in a few of the tubular lumens in the renal medulla of the experimental kidney. The proximal tubule, both convoluted and straight portions, was the most consistently damaged region of the nephron (Figs. 1, 2). The morphometric analysis of normal, injured, and necrotic proximal tubule cells in the outer, mid-, and cortical regions of the kidney at 3 and 24 hours after norepinephrine infusion is summarized in Table 1. The extent of injury was similar in all three regions of the cortex with damage occurring in both convoluted and straight portions of the proximal tubule. Renal tubular injury in the S2 regions within the straight portion of the proximal tubule could be easily quantitated; however, the cells of the S3 segment were more predisposed to sectioning damage so injury in this latter segment could not be reliably quantitated a t the 1-and 3-hour time intervals. Glomeruli did not show extensive alterations (Cronin et al., 1978). The amount of variation in the extent of injury among the animals was striking. In some animals, almost all of the proximal tubules were involved while in others there was little evidence of renal injury (Figs. 1, 2). In some instances, the major injury occurred in the proximal convolutions while in others it was localized to primarily the straight part of the proximal tubule. Renal lobules also frequently reacted differently with one having all proximal tubules injured while the adjacent lobule showed little evidence of damage. A layer of less injured tubules was also noted next to the renal capsule and surrounding thin-walled veins in the cortex. In this study, the contralateral kidney (noninfused) in each dog served as a control. These kidneys showed morphological features consistent with and indistinguishable from those of untreated mongrel dogs (Bulger et al., 1980).Both the 3- and 24-hour NE-infused kidneys had significantly greater tubular injury and necrosis compared to contralateral controls ( P < .001; Student’s t-test). 343 Stages of injury Injured proximal tubular cells tended to follow a definite progression through stages described as reversible injury to necrosis. These changes were similar to those described previously by Trump e t al. (1980)for the renal pedicle clamp model of ischemia. At 3 hours, paraffin sections showed mainly early changes such as apical blebbing (Fig. 3). The earliest changes seen by transmission microscopy involved distortions of the brush border with large cytoplasmic blebs being released into the lumens (Fig. 4). These blebs were generally free of formed organelles and sometimes appeared paler than the cytosolic density. Loss in the number and length of microvilli on the proximal tubular cells, simplification in cell shape, rounding of the mitochondria (Fig. 51, and clumping of nuclear chromatin characterized these early changes (Fig. 6). Intracristal swelling was not prominent. Red blood cells were seen within the interstitium (Fig. 6). The next alterations included vesiculation of the cytoplasm, which was especially prominent apically, a n early stage of matrical swelling of some of the mitochondria (Fig. 7) and dilation of the lumens of the endoplasmic reticulum (Fig. 8). A few of these mitochondria contained indistinct matrical densities (Fig. 9). The nuclei appeared more condensed a t this stage and the cells continued to simplify in shape. Some lateral cytoplasmic extensions extended out from the less injured cells to cover areas of the basal lamina which had already been denuded. The next stage was characterized by a decrease in cytoplasmic density, increased membrane vesiculation, and the presence of some mitochondria which had undergone high-amplitude swelling heralded the beginning of the transition to cell death (Fig. 9). Cells showing high-amplitude swelling of the mitochondria, marked discontinuities in the plasmalemma, membrane whorls, and a marked loss in cytosolic and nuclear density marked the conversion to necrotic debris (Figs. 9, 10). One-hour post norepinephrine The injury involved proximal tubules. The lumina contained material which appeared to be derived from blebbing of the proximal tubular cells. The proximal tubules in the outer stripe appeared to be obstructed with this material. The microvilli of the injured proximal tubules were distorted and disorganized. The apical cytoplasm in some proximal tubules displayed membrane vesiculation. Nuclear clumping was present. Aggregates of smooth endoplasmic reticulum were present. Although the majority of the injury was early and appeared to be of a reversible nature, some cells had progressed to a degree that injury was even identifiable in H & E sections. Occasional cells were necrotic, being characterized by mitochondria1 swelling with flocculent matrical densities. Three hours after norepinephrineinfusion Over half of the proximal tubules from outer, mid-, and inner cortex now demonstrated marked injury (see Table 1).A few necrotic cells were present at this time as well. The proximal tubules appeared expanded and filled with pink staining material. Some hyaline casts were present distally. Fig. 3.Light micrograph showing early changes in proximal tubules from dogs receiving an infusion of NE. The lumens are occluded with numerous cytoplasmic blebs. Hematoxylin and eosin, X 400. Fig. 5. Transmission electron micrograph showing the early proximal tubule alterations after NE infusion. There is loss in the number of microvilli on the apical surface and the cells are simplified in shape. ~4,000. Fig. 4.Transmission electron micrograph showing the early proximal tubule alterations after NE infusion. Note the large cytoplasmic bleb (B) which is present within the lumen. The brush border (BB) is markedly distorted. ~ 5 , 2 0 0 . Fig. 6.Transmission electron micrograph showing the early proximal tubule alterations after NE infusion. There is marked cytoplasmic vesiculation and clumping of the nuclear chromatin. A red blood cell has extravasated into the interstitium (arrow). ~ 4 ,5 0 0 . Fig. 7. Transmission electron micrograph showing early changes i n the proximal tubule. There is loss of brush border, a simplification i n the shape of the cell, increased cytoplasmic vesiculation, and early matrical swelling in the mitochondria. ~ 5 , 6 0 0 . Fig. 8 . Transmission electron micrograph showing t h e markedly dilated endoplasmic reticulum in proximal tubule cells after NE infusion. ~ 6 , 4 0 0 . Fig. 9. Transmission electron micrograph showing proximal tubule cells in various stages of reversible and irreversible cell injury. The cell a t the top of the micrograph shows clumping of the nuclear chromatin and increased vesiculation. The center cell shows more severe injury and marked mitochondria1 swelling is apparent (arrows). The cell in t h e lowcr portion of the micrograph demonstrates frank necrosis. There is dissolution of the cclular membranes and the mitochondria contain numerous flocculent densities. ~ 2 , 8 0 0 . Fig. 10. Transmission electron micrograph showing necrotic debris within the lumen o f t h e proximal tubule after NE infusion. Note the swollen mitochondria containing flocculent densities (MI and the membrane whorls (MW). ~ 7 , 2 0 0 . 346 R.E. BULGER ET AL. TABLE 1. Morphological analysis of normal, injured, and necrotic proximal tubules in norepinephrine-induced acute renal failure in the dog' Outer cortex (a) Normal NE3 3 hours' Postinfusion (N = 4) NE3 24 hours Postinfusion 42 rt8 41 +24 Injured Necrotic Normal Midcortex (%) Injured Necrotic Normal Inner cortex (%I Injured Necrotic 53 +8 5 +2 34 60 15 +7 6 *3 43 +6 +6 2 rt1 30 29 23 +20 33 38 *9 29 *23 37 +21 42 +8 + 17 + 10 55 21 'Values are means f SEM. Control dog kidneys exhibited less than 1%proximal tubule injury. 'The 3-hour postinfusion quantitation has been previously reported (Burke et al., 1984). 3NE = Norepinephrine. 24 hours after norepinephrine infusion Twenty-four hours after norepinephrine infusion, the amount of necrosis had increased as had the total number of tubules characterized by injury plus necrosis (Table 1). All zones of the cortex were equally involved. Hyaline casts were prominent in the lumen of distal nephron segments. Prominent margination of white blood cells was seen in the veins. Numerous red blood cells were seen within the cortical interstitium. DISCUSSION Although certain morphological changes have been mentioned in various studies (Cronin et al., 1978a; Schrier et al., 1978; Baehler et al., 1980; Cox et al., 1974) a complete description of the pattern and extent of injury has not been previously published. It is clear, however, that the ischemic injury seen after NE infusion differs markedly in distribution from the location described in the renal pedicle clamp model which has been carefully studied for the rat kidney (Glaumann and Trump, 1975; Glaumann et al., 1975a,b; Venkatachalam et al., 1978, 1981).With the renal clamp model in the rat, tubular injury occurs preferentially within the proximal pars recta. The production of acute renal failure (ARF) by the administration of NE has been described in several different species with varying doses and time intervals. Cox et al. (1974) induced irreversible ARF in dogs with a 2-hour infusion which was characterized by extensive tubular necrosis, principally involving the proximal convoluted tubules. They noted widespread loss or distortion of glomerular foot processes. The authors proposed that these glomerular changes were important in the decreased glomerular filtration rate found in this model. A subsequent study (Bulger et al., 1980) of the morphological changes seen in the glomerulus after the 2-hour NE infusion period noted only a slight increase in abnormal areas of podocytes but failed to find the extensive podocyte alterations reported by Cox et al. (1974). Cronin et al. (197813) produced a reversible model of acute renal failure in which norepinephrine was infused intrarenally for 40 minutes a t a dose of 0.75 pglkgl minute. The 40-minute norepinephrine infusion model has been carefully studied in elucidating the mechanisms involved in the production and maintenance phases of acute renal failure in the dog (Cronin et al., 1978a,b; Schrier et al., 1978; Burke et al., 1980, 1982; Patak et al., 1979; de Torrente et al., 1978).In the study of Cronin et al. (1978a), glomeruli of 37 of 39 animals appeared to have fairly normal structure while the proximal tubules were injured. In two other animals, more severe lesions characterized by necrosis of both proximal and distal tubules as well as glomerular changes similar to those reported by Cox et al. (1974) were seen. This reversible infusion model is useful since it demonstrates many of the characteristics seen in human acute renal failure such as a disproportionate decrease in glomerular filtration rate when compared to changes in renal blood flow, a decrease in the urine-to-plasma osmolality and creatinine ratios, a n increase in the urine sodium concentration, and a reversible course of azotemia. Normal glomeruli were described after a n 80-minute infusion of norepinephrine in dogs by Baehler et al. (1980). Taguma et al. (1980) has described a model of norepinephrine administration in which the length of the infusion was 30, 60, or 120 minutes in unilaterally nephrectomized dogs. In the early time periods studied in this model, blebs were released from injured proximal tubules and lodged in lumina of the tubules distally and suggested a pathogenetic role of tubular obstruction. No prominent glomerular foot process fusion was seen with any duration of infusion. Similar norepinephrine infusion studies were accomplished in rats by Steinhausen et al. (1978) and Conger et al. (1981). Tubules were flattened, dilated, and contained occasional casts. Glomeruli were described as normal after 90 minutes of NE infusion in the latter study. In the present 40-minute NE model the stages of tubule cell injury were similar to those described by Glaumann and Trump (1975), Glaumann et al. (1975), Donohoe et al. (1980), and Venkatachalam et al. (1978, 1981)for the renal clamp model. Intracristal swelling of mitochondria was never a prominent feature; however, early changes in luminal cell contours with apical blebbing were seen. The location of NE-induced injury did not show a preferential location to the proximal pars recta and was variable in degree and location. The injury occasionally could affect one lobule of the kidney while sparing a n adjacent one. In some kidneys, more injury was seen in the proximal convolutions while in others the injury centered on the proximal pars recta. The injury was more prominent in superficial nephrons in some instances while in others it was more prominent near the medulla. On average, however, the proximal tubules in all cortical regions of the kidney seemed to be involved to a similar degree (see Table 1).These differences between dogs and rats could relate in part to the NOREPINEPHRINE-INDUCED ACUTE RENAL FAILURE IN DOGS heterogeneous state of the mongrel dogs studied wherein age and previous medical and nutritional factors were unknown. This varying pattern of distribution correlates with the original description “remarkable patchy areas of renal ischemia” in humans by Oliver et al. (1951). It should be noted, however that necrosis of the more distal regions of the nephron, as reported by Oliver et al. (1951) for human ischemic acute renal failure, occurs with less frequency in experimental models and is generally seen only in the more severely injured kidneys (Cox et al., 1974; Cronin et al., 1978a). The norepinephrine model also differs from human acute renal failure because less cellular necrosis is evident in the human kidneys (Solez et al., 1979). One reason for this difference may be the fact that human biopsies are frequently taken several days after the initiating insult, as opposed to the immediate observation in the experimental model, and hence may reflect the reparative processes as well. As discussed above,.the majority of the studies of norepinephrine-induced injury clearly descibe no or minimal changes to the glomerular podocytes (Cronin et al., 1978a; Bulger et al., 1980; Taguma et al., 1980; Conger et al., 1981; Schrier et al., 1978; Baehler et al., 1980) unless severe irreversible damage has been sustained by the kidney as appears to be the case in the study of Cox et al. (1974) and with two of the 39 animals studied by Cronin et al. (1978a). In these instances, the podocyte changes may result from other factors such as proteinuria and not be the underlying cause of the decreased glomerular filtration rate seen in acute renal failure. On the basis of this and other morphologic studies (Taguma et al., 1980; Cronin et al., 1978a; Steinhausen et al., 1978; Baehler et al., 1980) it appears that the primary morphologic changes are injury and necrosis of the proximal tubules, which leads to nephron obstruction from cellular debris. Such tubular obstruction has been documented by the studies of Burke et al. (1980). The protective effect of agents which bring about increased osmolar excretion (Burke et al., 1980, 1983; Patak et al., 1979; Schrier et al., 1979; de Torrente et al., 1978) may be, a t least in part, a consequence of their ability to reduce tubular obstruction. In summary, the intrarenal infusion of norepinephrine to dogs produces a reproducible model of reversible acute renal failure. The extent and pattern of injury differ significantly from the renal artery clamping model of ischemia. ACKNOWLEDGMENTS The authors wish to thank Ms. Lena Wallach for her excellent secretarial assistance. This study was supported in part by National Institutes of Health grants 5-R01-AM-26134,AM-25151, and AM-19928. LITERATURE CITED Baehler, R.W., R.H. Williams, J. Work, J. Gotschall, and V. Chuang (1980) Studies on the natural history of the norepinephrine model of acute renal failure in the dog. Nephron 26:266-273. 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