Protection against ischemic acute renal failure by prostaglandin infusion

Prostaglandins Leukotrienes and Medicine 8:

361-373, 1982

PROTECTION AGAINST ISCHEMIC ACUTE RENAL FAILURE BY PROSTAGLANDIN INFUSION Anil K. Mandal, M.D. and Jon Miller, B.S. Section of Nephrology and Department of Medicine, Veterans Administration Medical Center and University of Oklahoma, Oklahoma City, Oklahoma 73104 ABSTRACT We have shown that intravenous infusion of epinephrine (4ug/kg/min for 6 hours) into mongrel dogs consistently produces renal hemodynamic and histopathologic characteristics of ischemic acute renal failure (ARF). This study describes renal responses that were modified by intravenous infusion of prostaglandin E (PGE )(lO u /min) one hour before and during a 6 hour infusion of epine6hrlne ’ ?4 ug/kg/min). Two groups of animals were studied: Group I (epinephrine alone) and Group II (epinephrine + PGE ). Urine volume, glomerular filtration rate, urinary sodium excretii on rate, urine osmolality, and serum urea nitrogen were measured. Renal tissues were studied using light and electron microscopy. While urine volume or glomerular filtration rate decreased significantly in both groups, they were slightly but significantly better in Group II than Group I. Urine osmolality significantly decreased in Group I but significantly increased in Group II. Group I animals became azotemic (mean serum urea nitrogen, 27 + 1 mg/dl), whereas Group II animals showed serum urea nitrogen at the upper limits of normal (mean 20 + 2 mg/dl). The difference was significant (P c.01). Severe acute tubular lesions were a consistent feature in Group I. Tubular lesions were less severe and infrequent in Group II animals. While mitochondrial dark bodies (electron microscopy) characterized tubular lesions in Group I, fewer mitochondria contained dark bodies in Group II animals. These dark bodies appear to be calcium and constitute a definitive sign of ischemia. Therefore, this study indicates that PGE attenuates epinephrine-induced tubular ischemia and injury and ARF whl-6h may be attributed to excessive solute excretion or to inhibition of calcium influx into tubular mitochondria. This work has been supported in part by the Medical Research Service of the Veterans Administration and in part by a Grant (No. ROlAM26022-02) from the National Institute of Arthritis, Metabolic, Digestive and Kidney Diseases. 361

INTRODUCTION Acute renal failure (ARF) occurs in an indeterminate percentage of patients following massive loss of blood or fluid, massive trauma, This ARF is endotoxemia, prolonged surgery, or obstetric accidents. accompanied by a 12-50 percent mortality despite dialytic therapy (1). Although hypovolemia, hypotension, or myoglobinuria has been incriminated as the immediate cause of ARF, exact pathogenetic Decreased prostaglandin activity mechanisms still remain undetermined. has been implicated in the pathophysiology of ischemic ARF (2). Though intrarenal or intravenous infusion of prostaglandin E (PGE ) has been shown to provide partial renal hemodynamic or histopatholo 6.lc protection against ischemia or glycerol-induced ARF; the pathway(s) of this protection has not been delineated (3-5). We have shown that intravenous infusion of epinephrine (4 ug/kg/min for 6 hours1 in mongrel dogs produces acute tubular necrosis (ATN) with oliguria and azotemia, and chronic splenectomy protects against this ARF (6-7). This study was initiated to examine whether infusion of exogenous PGE can prevent the development of epinephrine-induced ARF and aid.in unde 6 standing how renal protection can be afforded by this vasodilator agent. MATERIALS AND METHODS Seventeen mongrel dogs of 18-20 kg body weight were studied. They were observed for a week to ensure a disease free state and divided into Group I - Epinephrine alone (n = 7); Group II - Epinephrine two groups: + PGE2 (n = 10). All animals were anesthesized with sodium pentobarbital (30 mg/kg body weight). In all animals, one femoral artery was catheterized to monitor arterial pressure (Statham transducer, Statham Instruments, Inc. Oxnard, California), and both femoral veins were catheterized to deliver a solution of epinephrinf2,$Epi) through one vein and a solution of PGE2 and sodium iothalamate I (Glofil 125, Abbott Laboratories, North Chicago, Illinois) through the other vein. In three Group II animals, a catheter was placed into the left ventricle.. The bladder was exposed through a suprapubic incision, both ureters were identified and catheterized with PE tubing 190, 3-4 cm proximal to the bladder, and connected to a container for the collection of urine. In each animal, the left kidney was exposed through a flank incision, and the renal artery was cleared and separated from the remaining structures. An electromagnetic flow transducer was placed around the renal artery as close to the kidney as possible and connected to a Micron 1000 C flow meter functioning through a Sanborn polygraph recorder. Urine was collected hourly before and during infusion of Epi and blood was drawn from the arterial line at the mid-point of each urinary collection. For t&5c011ection of control samples, a priming dose of sodium iothalamate I (10 UC) was given intravenously as a bolus. Normal saline solution was then infused at the rate of 2 ml/min for one hour to maintain urine flow. After the one hour priming periof2bpi (4 ug/kg/min), and a sustaining dose of sodium iothalamate I (.Ol uC/min) were delivered in normal saline form two separate bottles via a peristaltic pump at the rate of 1 ml/min each for 6 hours. A stock solution of PGE (purchased from Upjohn Pharmaceutical Company, Kalamazoo, Michigan) wa P prepared in

362

absolute ethanol and stored at -20' C. Just prior to the experiment, the stock solution was diluted with normal saline to provide an infusion of 10 ug/min PGE2 into Group II dogs during the control hour and 6 hours of Epi infusion. It is well known that PGE is degraded by the lungs (8), therefore, the amount of PGE circulat?ng in the kidneys after intravenous infusion remains unpr$dictable. To obviate pulmonary degradation in three Group II animals, PGE, was infused directly into the left ventricle to ensure at least one i; ass of PGE at the rate of 10 ug/min through the kidneys (these three animals did n6t receive PGE through femoral vein). The total volume of fluid infused in each ai;. imal remained constant (2 ml/min) before and during infusion. Mean arterial blood pressure was monitored continuously and renal blood flow was recorded hourly. At the end of the six hour period, the animals were sacrificed. From each animal, the right kidney was removed, a wedge from the cortex was fixed in 10 percent buffered formalin for light microscopy (LM), and a few 0.5 to 1 rrmpieces from the cortex were fixed in 4 percent buffered glutaraldehyde (pH 7.4) for transmission electron microscopy (EM) studies. Hematocrit, and serum urea nitrogen (SUN) were measured at 0, 1, 3, and 6 hours. Urinary samples were measured for volume, osmolality12gnd sodium concentration. Blood and urine samples were analyzed for I concentrations and glomerular filtration rates were determined using standard formula. Urine osmolality was determined by freezing point depression with an osmometer (Fiske Associates). Urine sodium concentration was measured by flame photometry (Instrumentation Laboratories). Sodium excretion rates were determined by multiplying sodium concentration by urinary volume per minute. Serum urea nitrogen (urease method) was determined by the Automatic Beckman Astra. HISTOPATHOLOGIC STUDIES The preparations and techniques for LM and EM studies were similar to those in our previous studies (9). LM. and EM evaluations were made blindly without prior knowledge of the type of experiment. Acute tubular lesions (ATL) were graded on a scale of 0 to 4+ and renal congestion was graded on a scale of l+ to 3+ in order of increasing severity (9). STATISTICAL METHODS All the data were analyzed with reference to differences among control (0) hour and l-6 hour infusion periods within each group and between the two groups. Mean values + SEM were used in the presentation of data and a student's "t" test, P < .05 was utilized for determining significant difference. RESULTS RENAL FUNCTION STUDIES Urine volume (UV), sodium excretion rate (UNaV), urinary osmolality (Uosm), and glomerular filtration rate (GFR) are shown in Figure 1.

363

0

hrs

6

1

URINE

OSMOLALITY

SODIUM

601,

.31T

hrs

URINARY EXCRETION

0

6

1 URINE

?? GR oGR

hrs

0

1

6

GLOMERULAR FILTRATION RATE

VOLUME

I II

Renal Function studies. Top panel: urine osmolality sodium excretion (_UNaV). There was no difference in Uosm at 0 (Control) or during 1 hr Epi infusion. At 6 hr, Uosm was significantly different between two groups; UNaV was not different at 0 hr between groups, at 1 hr UNaV was significantly higher in Group I than Group II. Thereafter, UNaV decreased in Group I but increased in Group II and at 6 hr the difference was significant. Bottom panel: Urine volume (UV) and glomerular filtration rate (GFR). No difference was found in UV at 0 hr and 1 hr but at 6 hr UV was significantly different between groups; GFR showed similar patterns like UV between two groups before and during infusion of Epi. FIGURE

1:

(Uosm)nd

364

TABLE I Hematocrit (Vol %) Mean * SEM Hours Group I (Epi only) Group II (Epi + PGE2)

*P

< .05;

39:1 3753

Epi = Epinephrine

53:2 45*5*

3 63+1 52+4*

6222 53*3*

0 = Control

TABLE II Grades and Scores of Acute Tubular Lesions (ATL) a Renal Congestion Grades

Group I (n = 7)

ATL

Group II

(n = 7)

0

1+ 2+ :I Mean Score + SEM

: 0

2” 0

; 3.3 .2

Fl 1.1* .5

RENAL CONGE,STION

:r

k!

3+ Mean Score * SEM

*P **p

< <

257 . 7-

.Ol .05

365 PAM-

C

; 2 1.9** .3

Since there was no difference found in the renal hemodynamic or histopathologic parameters between the animals that received PGE intravenously and those given intraventricular infusions, the da?a obtained from seven animals infused with PGE are pooled and presented. There was no difference in any of the renal ? unction parameters between Group I and Group II at control (0) hour. During sixth hour infusion period, there were significant decreases in all renal function parameters in Group I compared to those in Group II. These differences are Group I vs Group II: UV (.03 ? .03 vs .12 + .02 ml/min; P < .05); GFR (6 f 3 vs 20 f 4 ml/min; P .05); UNaV (1 + .4 vs 7 i 2 uEq/min; P ( .05); Uosm (444 + 43 vs 1220 i 190 mosm/kg; P c .OOl). Urinary potassium excretion rate, like UNaV, was significantly (P < .OOl) lower in Group I (2 + 1 uEq/min) than Group II (13 ? 2 uEq/min). There was no difference observed in renai blood flow before and during infusion between the two groups. Though SUN was elevated at 6 hour in both groups the degree of elevation was significantly greater in Group I than Group II (27 it- 1 vs 20 + 3 mg/dl; P / .05). Mean arterial blood pressures (MAP) at 1 hr (125 i 9 min Hg) and 6 hr (65 + 28 mm Hg) were significantly lower (P < -05) in Group II than in Group I. The MAP in Group I at 1 hr and 6 hr were 183 i 10 mm Hg and 130 ? 8 mm Hg, respectively. The hematocrit results are provided in Table I. Hematocrit increased significantly during infusion in both Group I and Group II. However, hematocrit levels at all hours during infusion were significantly lower in Group II than in Group I. HISTOPATHOLOGIC

STUDY ,

LIGHT MICROSCOPY The grades and scores of ATL and renal congestion have been presented in Table II. All seven Group I dogs sustained 3+ to 4t ATL (Figure 2). Tubular lesions were comparatively less severe in Group II than in Group I. Among Group II dogs, three dogs had no ATL (Figure 3), two dogs had l+ ATL, and two dogs .had 3+ ATL. The severity score of ATL (1.1 + .5) was significantly lower (P < .Ol) in Group II than in Group Renal congestion was marked in Group I and significantly < .05) less severe in Group II animals. tP(3*3 k .2). ELECTRON MICROSCOPY Severe tubular changes were observed in Group I animals. These changes were characterized by swelling of the mitchondria along with disorganization of the cristae and appearance of large dark bodies, disruption and displacement of microvilli, and presence of necrotic epithelial cells within the lumina (Figure 4). These ultrastructural findings contrasted with those in Group II in which tubules revealed subtle changes such as, a slight separation of the tight junctions of the cells, a few vacuoles, and small dark bodies in a few mitochondria (Figure 5). A quantitative analysis of the mitochondrial dark bodies show 48 - 53 percent mitochondria contained large dark bodies in Group I while less than 25 percent mitochondria contained small dark bodies in Group II animals.

366

FIGURE 2; Light microscopy of the kidney from a Group I dog reveals collapse of the glomerular tufts with enlargement of the Bowman's space (6s) and diffuse necrosis of tubules (T). In many tubules, basement membranes are disrupted as shown here (arrowheads). Complete detachment of epithelium and cast formation within a tubule seen (C). Tubules that are not necrotic show flattening of the epithelium with wide lumina OJ,,,;; general, nucle? of all tubules are inconspicuously . Peritubular ca illaries are filled with red blood cells (congestion)(arrowsP. H&E x 120

367

FIGURE 3: Light microscopy of the kidney from a Group II dog reveals a normal appearing glomerulus and essentially normal tubules. A few distal tubules are slightly dilated (DT). Nuclei of all tubules stand out. Peritubular capillaries are devoid of congestion. H&E x 120

368

FIGURE 4: Electron microscopy of a proximal tubule from a Group I dogals severe changes in the mitochondria, characterized by swelling, disappearance of the cristae, and appearance of large dark bodies (_circles). These dark bodies appear to be located at the edge of the mitochondria. There is a conspicuous absence of plasma membrane infolds and cytoplasmic constituents. Cellular cytoplasm is edematous and contains lipid droplets (L). Condensation of chromatin in the nucleus (.N)and fragmentation of microvilli (MV) are shown. UA + LC x 8,000

369

FIGURE 5: Electron microscopy of a proximal tubule (5 segment) from a Group II dog demonstrates preservation of the c&lular constituents such as Golgi complexes (-arrowhead)in most tubular cells. Plasma membranes (single arrows) and microvilli (MV) are generally intact. Except showing small dark bodies in a few mitochondria (circles) most mitochondria appear essentially normal. Nucleus (N) shows prominent nucleolus and no condensation of chromatin. UA + LC x 7,000

370

DISCUSSION This study demonstrates that intravenous infusion of a high dose of PGE (10 ug/min) along with epinephrine provides a significantly imp?oved renal function and a histomorphologic protection compared to animals infused with epinephrine alone. Other investigators have found a significantly lower serum creatinine in rats treated with PGE (3.4 Umol/L) along with glycerol than in rats which received glycero? alone. However, acute tubular necrosis (ATN) was not averted (5). Still other investigators have found that infusion of PGE intrarenally before and during intrarenal infusion of norepinephrine 8ecreased renal blood flow similar to that during norepinephrine alone, but inulin clearance (GFR) was reduced to a significantly lesser extent three hours after discontinuing norepinephrine (3). From all these studies, it is evident that exogenous PGE2 subserves a protective function against a variety of ARF. The mechanism of renal protection afforded by PGE has not been clearly delineated in the previous studies. We have m0de two observations in PGE infused animals: high urinary osmolality and a reduced number of m ?tochondrial dark bodies in the proximal tubules which may be relevant in explaining the PGE protective effect. First, it has been proposed that an increase in soPute excretion may be important in the renal protection by various agents. Thus, bradykinin and furosemide, which increase renal blood flow as well as urine flow and solute excretion, have been shown to provide renal protection against norepinephrine, while, in contrast, secretin, which increases renal blood flow without altering solute excretion, has no protective effect in the norepinephrine model (10). Similarly, infusion of hypertonic mannitol causes a shrinking of tubular cell swelling with regression of ATN and slight but significant improvement of renal The authors explained that function in an ischemic ARF model (11). mannitol, being nonabsorable, counterbalanced the osmotic effect of intracellular solutes and prevents or reverses cell swellin (11). It has been reported that PGE increases osmolar clearance (127 Therefore, the excessive &lutes excreted (higher Uosm) in PGE infused animals (Group II) found in our study might in some way have p?eserved the normal morphology of the tubular epithelial cells and prevented complete shutdown of renal function. Second, mitochondrial dark bodies are a distinct sign of ischemia, the larger the size of dark bodies, the more severe is the ischemia (13). Thus, the EM study, showing many mitochondria containing large dark bodies (Figure 4) has provided definitive evidence of severe ischemia in the tubules of Epi infused animals. Conversely, fewer mitochondria containing small dark bodies imply mitigation of ischemia in the tubules of PGE infused animals. These dark bodies appear to be calcium (14). Cal&m influx into the tubular cells, producing ATN and ARF, remains a possibility, especially since verapamil, a calcium blocker, has been shown to attenuate norepinephrine-induced ARF (15). How PGE2 can minimize mitochondrial calcification (dark bodies) is not clear. It has been reported that excitable cells (such as after e inephrine) possess a slow inward calcium current (calcium channel7 . This calcium chanel is activated by the interaction of beta adrenergic agonists (such as epinephrine); this latter effect may involve mediation by elevated cyclic AMP levels (16).

371

Since PGE has been shown to inhibit cyclic AMP formation (17), it is possible hat PGE has attenuated calcification of the tubular mitochondria ? by a + fecting this calcium channel (influx). A significantly lesser increase in hematocrit and significantly less severe renal congestion may be attributed to decreased splenic contraction. Decreased release of norepinephrine from the spleen following PGE infusion has been observed (18). Since splenic function has been impl?cated in the pathogenesis of epinephrine-induced ARF (6, 7, 9) decreased splenic function in attenuating the severitv of ARF following PGE2 infusion remains a possibility. Trauma, prolonged surgery or shock are known to cause an excessive outpouring of catecholamines (19). These conditions are frequently associated with ARF, which is similar to the ARF produced by an in vitro infusion of epinephrine in our model. Therefore, the information derived from this model, with regard to the beneficial effect of PGE infusion in ischemic ARF, should stimulate further investigations fo ? the potential application of PGE2 in clinical acute renal failure. ACKNOWLEDGEMENTS The authors are grateful to Dr. Lerner B. Hinshaw, Department of Surgery for providing a part of the funds in purchasing PGE . We wish to express appreciation to Mrs. Alvia M. Woodfork, Nephrolo i; y Section for the secretarial assistance in the preparation of this manuscript. REFERENCES

1)

Kleinknecht, D., Jungers, ., Chanard, J., Barbanel, C., Ganeval, D.: and Roudon-Nucete M. Factors Influencing immediate prognosis in acute renal failure with special reference to prophylactic hemodialysis. Advances in Nephrology (J. Hamburger J. Croshier, and MH Maxwell, eds) Year Book Medical Publisher, Chicago. 207, 1971.

2)

Conger, J. D., and Schrier, R. W. Renal hemodynamics Ann Rev Physiol 42: 603, 1980. failure.

3)

Mauk, R. H., Patak, R. V., Fadem, S. Z., Lifschitz, M. D., and Stein, J. H. Effect of prostaglandin E administration in a nephrotoxic and a vasoconstrictor model of acute renal failure. Kidney Int 12: 122, 1977.

4)

Casey, K. D., Machiedo, G. W., Lyons, M. J., Slotman, G. J., and Novak, R. T. Alteration of post-ischemioc renal pathology prostaglandin infusion. J Surg Res 29: 1, 1980.

5)

Werb, R., Clasrk, W. F., Lindsay, R. M., Jones, E. 0. P., Turnbull, D. I., and Linton, A. L. Protective effect of prostaglandin (PGE ) in glycerol-induced acute renal failure rats. Clin Sci MO 7 Med 55: 505, 1978.

372

in acute renal

in

by

6)

Bell, R. D., Mandal, A. K., and Parker, D. E. The effect of splenectomy on renal function in epinephrine-induced renal failure. Proc Sot Exp Biol Med 167: 12, 1981.

7)

Mandal, A. K., Llach, F., Miller, J., Nordquist J., Fahmy, A., Patak, R. V. and Wilson, M. F. Chronic splenectomy protection against epinephrine-induced acute renal failure in dog. (Submitted.)

8)

Mookerjee, B. K. and Patak, R. V. Vasodepressor substances extractable from kidney tissue in the Renal Papilla and Hypertension, (Eds: Mandal, A. K. and Bohman, S. (.), Plenum Press, N. Y., N. Y. 77, 1980.

9)

Mandal, A. K., Haygood, C. C., Bell, R. S., Sethney, T. James, T. M., Nordquist, J. A., Yunice, A. A., and Lindeman, R. D. Effect of acute and chronic splenectomy on experimental acute renal tubular lesions. J Lab Clin Med 92: 698, 1978.

10)

Patak, R. V., Fadem, S. A., Lifschita, M. D., Stein, J. H. Study of factors which modify the development of norepinephrine-induced acute renal faiure in the dog. Kidney Int 15: 227, 1979.

11)

Frega, N. S., Dibona, D. R., and Leaf A. Enchancement of recovery from experimental ischemic acute renal failure. Renal Pathophysiology (Eds: Leaf et al.) Raven Press, New York, 203, 1980.

12)

Lee, J. B., Patak, R. V., and Mooerjee, B. K. Renal prostaglandins and the regulation of blood pressure, sodium and water homoestasis. Am J Med 60: 798, 1976.

13)

Jennings, R. B., Baum, J. R., and Herdson, P. B. Five structural changes in myocardial ischemic injury. Arch Path 79: 135, 1965.

14)

Hagler, H. K., Sherwin, L., Buja, M. Effect of different methods of tissue preparation of mitochondrial inclusion of ischemic and infarcted canine, myocardium. Lab Invest 40: 529, 1979.

15)

Burke, T. J., Arnold, P. E., Grossfield, P. D., and Schrier, R. W. Effect of calcium membrane inhibition on norepinephrine-induced acute renal failure. Abstract Book. The Tel Aviv Satellite Symposium on Acute Renal Failure, 108, 1981.2

16)

Gevers, W. 406, 1981,

17)

Geza, F-T, Magyer, A. and Walter, J. Renal response to vasopressin after inhibition of prostaglandin synthesis. Am J Physiol 232: F416, 19077.

18)

Hedqvist, P. Control by prostaglandin E of sympathetic neurotransmission in the spleen. Life Sciencgs 9: 169, 1970.

19)

Lillehei, R. C. History of vasodilation in treating shock an low-flow states. Advances in Shock Research, Vol. I (Eds. lefer et al.), Alan R. Liss, New York, 1, 1979.

Calcium:

The managing director.

373

S Afr Med J 59: