Epidermal growth factor accelerates renal repair in mercuric chloride nephrotoxicity TEREZILA M. COIMBRA, DEBORAH A. CIESLINSKI, Departments of Internal Medicine, Veterans Administration and University of Michigan, Ann Arbor, Michigan 48105
M., DEBORAH A. CIESLINSKI, AND H. growth factor accelerates renal repair in mercuric chloride nephrotoxicity. Am. J. Physiol. 259 (Renal Fluid Electrolyte Physiol. 28): F438-F443, 1990.-Repair and recovery of ischemic or nephrotoxic acute renal failure (ARF) are dependent upon renal tubule cell regeneration. Because epidermal growth factor (EGF) is a potent growth promoter to renal tubule cells, experiments were undertaken to assess the effects of exogenous administration of EGF during the recovery phase of HgClz-induced ARF. Rats were administered HgCl, (5 mg/kg SC), and [“Hlthymidine incorporation into renal tissue and blood urea nitrogen (BUN) and serum creatinine concentrations were measured at various times after toxin administration. EGF (20 pg) was administered subcutaneously 2 or 4 h after HgCl, injection. Exogenous EGF resulted in greater levels of renal [‘Hlthymidine incorporation into renal proximal tubule cells compared with those observed in nontreated animals at several time points in the first 48 h after toxic injury. Morphometric analysis of histoautoradiograph sections of renal tissue demonstrated that >96% of labeled cells were tubular in all examined sections. This EGF-related acceleration in DNA synthesis was associated with significantly lower peak BUN and serum creatinine levels, averaging 213 t 23 and 6.54 t 0.72 (SE) mg/dl, respectively, at 3 days in EGF-treated nephrotoxic rats compared with peak levels of 359 t 40 and 9.92 t 1.67 mg/ dl (P < 0.001, n = 7-16) at 5 days in non-EGF-treated nephrotoxic rats. EGF treatment also was associated with a return to near normal BUN and serum creatinine levels approximately 4 days earlier than that observed in non-EGF-treated animals. These findings demonstrate that exogenous EGF accelerates the repair process of the kidney after a severe toxic insult. COIMBRA, TEREZILA DAVID HUMES. Epidermal
acute renal failure; tubule cell regeneration; renal recovery; deoxyribonucleic acid synthesis; thymidine
function
AND H. DAVID Medical Center,
HUMES
Epithelial cells at both ends of the necrotic segments of proximal tubules, as well as other surviving epithelial cells along the denuded surface of the tubular basement membrane, appeared to be responsible for the regenerating cells. In this manner most tubules were relined with new epithelium by day 5. More recent work has demonstrated a time course similar to that observed with HgC1, for renal tubule cell regeneration and renal function recovery after bilateral renal ischemia in the rat (10). These observations suggest that local factors play a significant role in mediating the regeneration process leading to recovery from acute renal failure. This process of regeneration and repair by surviving epithelial cells at the edge of the injury segment is likely dependent on either autocrine or paracrine production of growth-promoting factors. Several growth factors, including platelet-derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor (TGF)-~tl and TGF-P have all been shown to be important in wound healing and reparative processes (20). Of these growth factors, EGF is one of the most potent mitogenic stimuli of renal proximal tubule cells (9, 16). In this regard, a recent report has documented that exogenous administration of EGF early in the recovery phase of ischemic acute renal failure accelerated epithelial cell replication and condensed the time required for renal function recovery (10). This series of studies was therefore carried out to determine whether exogenous EGF has a similar effect of accelerating renal tubule cell regeneration and renal repair in a nephrotoxic model of acute renal failure induced with HgCl,. METHODS
or toxic acute renal failure is a reversible form of organ failure. It has both an injury and recovery phase. The injury phase is due to structural damage to renal epithelial cells, predominantly proximal tubule cells (12). The recovery phase is therefore dependent on the repair and replacement of the injured and necrotic tubule epithelial cells. A classic report carefully evaluated renal epithelial cell regeneration after mercuric chloride (HgCl&-induced toxic acute renal failure in the rat (7). This toxic event resulted in necrosis of proximal tubule cells during the first day after HgCl,. Regeneration of epithelial cells, as reflected by renal thymidine incorporation and radioautography, began by day 2 with immature epithelial cells relining the necrotic areas by day 3. ISCHEMIC
F438
0363-6127/90 $1.50 Copyright
0
Male Sprague-Dawley rats (250-325 g) were administered HgCl, (5 mg/kg SC) followed at 2 or 4 h with a single subcutaneous injection of EGF (20 pg) or sham vehicle (0.9% NaCl). At time intervals of 24, 30, 48, and 72 h after toxin administration, [3H] thymidine incorporation into renal cortex, as well as blood urea nitrogen (BUN) and serum creatinine levels, were determined with methods detailed previously for this laboratory (10, 11). To obtain a complete time course for BUN and serum creatinine levels, some animals were allowed to recover for as long as 12 days after HgClz administration, and blood samples were obtained daily for BUN and serum creatinine levels. To localize and identify the cells incorporating the
1990 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 31, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
EGF
AND
TOXIC
ACUTE
radiolabeled thymidine within the kidney, histoautoradiography of kidney samples was also accomplished by use of methods previously published by this laboratory (10,ll ). Sections were examined at x 176 magnification, and 20-30 fields were counted in renal cortex from an individual animal. The cell type of the labeled cells was identified as either tubular or interstitial. To determine the number of tubule cells incorporating [3H]thymidine after various treatment regimens, 10 random fields in renal cortex and outer medu lla were selected from histoautoradiograph s of kidney specimens, and labeled tubule cells were counted in each field. To ensure that EGF administration did not affect toxin delivery to the kidney and therefore the degree of tubule cell injury, quantitative assessment of morphological alterations induced by HgCl, was accomplished with light microscopy of hematoxylinand eosin-stained sections. By use of a blinded quantitative grading scale previously published by this laboratory (22), the amount of injury in 20 proximal tubules in the inner cortex and 20 proximal tubules in the outer stripe of the outer medulla were graded from 0 ( completely normal tubule) to 5 (maximal loss of tubular architecture with extensive loss of tubular cells). After centering either inner cortex or outer stripe in a low-power field, the high-power objective was positioned to view a field at random. Each field usually contained four or five proximal tubules. If the field had more than five proximal tubules, then the five tubules most central in the field were graded. All reagents used were of the highest grade commercially available. All organic reagents were obtained from Sigma Chemical (St. Louis, MO) unless otherwise indicated. [MethyL-3H] thymidine was obtained from New England Nuclear (Boston, MA). EGF was obtained from AmGen Biologicals (Thousand Oaks, CA). Statistical analysis was performed by use of either Student’s t test or analysis of variance (ANOVA) to identify significant differences between treatment groups. If significant differences between treatment groups were identified by ANOVA, specific comparisons between individual treatment means were accomplished using Tukey’s multiple comparison procedure in conjunction with the Studentized range critical values table. RESULTS
The study design produced a model of reversible nephrotoxic acute renal failure, as demonstrated in Fig. 1. Renal excretory function declined quickly following HgCl, injection, as reflected by BUN levels rising in the first 24 h from an average (&SE) control level of 21 t 2 to a mean of 96 t 4 mg/dl (n = 32). BUN levels peaked to levels of 359 t 40 mg/dl (n = 7) at day 5 after toxic insult followed by progressive improvement thereafter to near normal levels by day 12. As an indication of increases in cell regeneration, [3H]thymidine incorporation into DNA of the kidney began to rise as early as 24 h with dramatic increases between 24 and 48 h after toxic injury (Table 1). Histoautoradiographic studies were performed to determine the type of cells labeled with [3H] thymidine after HgCl, exposure (Fig. 2). Kidney sections from rats at 48 or 72
RENAL
F439
FAILURE
HgCI, -___+ + _---EGF
-
E
:? : I :: * :, : :+..., *** ‘* T------,.-* ** * * * --a--,-, * --~------)-----~------
200
Z 3
m 100
0' 0
12
3
4
5 Time
6 7 (Days)
8
1
9101112
FIG. 1. Time course for blood urea nitrogen (BUN) values in rats administered HgClz at day 0. The group of animals receiving a single dose of epidermal growth factor (EGF; 20 pg) within 2-4 h of HgCl, injection had BUN levels significantly lower at various time points than the animals administered HgCl, not treated with EGF (* P < 0.05, ** P c 0.01, *** P < 0.001, 72 = 5-33).
h after toxic administration were used for histoautoradiographic analysis. Labeling was confined to the nuclei. On average, 97.9% of the labeled cells were tubule cells, and 2.1% of the labeled cells were interstitial cells in the 60 examined fields. In control rats undergoing sham injection, 98.4% of labeled cells were tubule, and 1.6% were interstitial in 90 examined fields. A single dose of exogenously administered EGF at 2 or 4 h after HgCIZ resulted in higher rates of renal thymidine incorporation compared with values observed in non-EGF-treated animals receiving the nephrotoxin at 24, 30, 48, and 72 h (Table 1). When the EGF treatment group was compared with the nontreated group, the effect of EGF to increase renal thymidine incorporation was highly significant by ANOVA (P = 0.0011). Thus EGF significantly enhanced renal thymidine incorporation during the period of peak replicative and repair response of renal tubule cells to HgClz-induced cell necrosis. Of note, EGF treatment of control rats not receiving HgC12 did not demonstrate any increase in [3H]thymidine incorporation in kidneys. At 24 h and after a single dose of EGF in rats undergoing sham HgClz injection, renal [3H]thymidine incorporation averaged 13 t 4 x lo3 disintegrations per minute (dpm)/mg DNA, compared with a value of 12 t 2 X lo3 dpm/mg DNA in nonEGF-treated sham animals (P = NS, n = 4). Histoautoradiographic studies were performed to determine the location of the cells labeled with [3H]thymidine in kidney sections from rats treated with EGF at 48 h after HgCl, injection. Labeling was again confined to nuclei. On average, 96.3% of labeled cells were identified as tubule, and 3.7% labeled cells were identified as interstitial cells in 60 fields. To test the effect of EGF on renal tubule cell replication after HgClz-induced injury in a more absolute manner than measurement of the specific activity of renal [3H] thymidine (Table l), the number of labeled cells in renal cortex and outer medulla was determined at 24 and 48 h after HgC12 administration in both treatment groups. At 24 h EGF treatment increased the number of tubule cells incorporating [3H] thymidine, as assessed by
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 31, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
F440 TABLE
EGF AND TOXIC
ACUTE
RENAL
FAILURE
1. Effect of EGF on renal thymidine incorporation after HgC12 [3H]Thymidine Incorporation, dpm Oh
24 h
30
x
104/mg DNA
h
48
h
12
h
0.13rto.04 HgCl, 8.42+1.08 20.37k4.24 64.16k7.59 99.80+28.04 HgClz + EGF 0.12&0.03 13.81&1.28* 42.03k5.45t 80.52k19.52 116.24+13.80 Values are means + SE; n = 4-8 animals for each group. Time course for renal thymidine incorporation in rats administered 5 mg/kg HgC12. Epidermal growth factor (EGF) was administered 2-4 h after nephrotoxin administration. Relative to non-EGF treatment group, * P < 0.02 and t P < 0.005 by one-way ANOVA and Tukey’s multiple range procedure.
FIG. 2. Histoautoradiography of renal cortex of a kidney from a rat 72 h after HgCl, injection. [3H]thymidine label is observed as black-silver grains above tubule cell nuclei (magnification x500).
histoautoradiography, from 1.6 f 0.4 in the nontreated groups to 7.5 + 1.8 labeled cells/field in the treated group (P < 0.02, n = 3 rats and 60 fields for each group). Similarly at 48 h, EGF increased labeled tubule cells from a nontreated value of 12.1 & 1.8 to 20.9 + 2.1 cells/ field (P < 0.01, n = 3 rats and 60 fields for each group). Thus, EGF significantly enhanced renal tubule cell replication in the early reparative phase after nephrotoxic injury as assessedboth by specific activity of [3H]thymidine in the kidney and by absolute numbers of renal tubule cells incorporating labeled thymidine into DNA. Associated with this EGF-induced enhancement of tubule cell thymidine incorporation, the time course of renal excretory failure after nephrotoxic injury, as measured by both BUN and serum creatinine levels, was dramatically altered by EGF treatment as demonstrated in Figs. 1 and 3. EGF-treated animals had significantly lower peak BUN and serum creatinine levels, averaging 213 rt 23 and 6.54 + 0.72 mg/dl, respectively, at 3 days after HgClz injection, compared with peak levels of 359 + 40 and 9.92 -I- 1.67 mg/dl (P < 0.001, n = 7-16) at 5 days in nontreated nephrotoxic rats. EGF treatment also was associated with a return to near normal BUN and serum creatinine levels in EGF-treated animals on day 8,4 days earlier than that observed in non-EGF-treated animals. EGF treatment did not alter the degree of Hg2+ presented to the kidney, because no difference in histological
HgC’z
I2 r
HgClp. . . +. . EGF
-
0
2
4
5
Time
8
10
(days)
FIG. 3. Time course for serum creatinine values for animals with HgClz-induced acute renal failure. Animals treated with epidermal growth factor (EGF) had significantly lower (* P < 0.05, ** P < 0.01, n = 4-16) values compared with untreated rats with toxic renal injury.
score of tubule cell injury was demonstrated between the nephrotoxic rats treated or not treated with EGF. Histological grading was assessedat 24 h, a time at which the peak of HgCl,-induced toxic renal injury occurs and just before the accelerated regeneration repair phase (6, 7, 13). At 24 h, the mean histological scores in the inner cortex and outer stripe were 2.92 + 0.13 and 4.70 + 0.07, respectively, in the nephrotoxic non-EGF-treated rats vs. scores of 3.25 rt 0.18 and 4.65 & 0.08, respectively, in the nephrotoxic EGF-treated rats (n = 120 fields from 3
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 31, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
EGF AND TOXIC
ACUTE
RENAL
FAILURE
F441
FIG. 4. Representative light micrograph of inner cortex (x90) from rat 24 h after HgCl, administration. Extensive tubule necrosis is present with necrotic debris filling tubular lumina.
animals in each treatment group, P = NS for both scores). As depicted in Figs. 4 and 5, at 24 h after HgCL, extensive necrosis was present in proximal tubule segments with no difference in the degree of tubule cell necrosis observed histologically between the EGFtreated and nontreated groups receiving HgCL DISCUSSION
Because acute renal excretory failure from nephrotoxic or ischemic stress is due to segmental tubule cell necrosis, the reversibility of nephrotoxic or ischemic acute renal failure is dependent upon renal epithelial cell regeneration to reconstruct normal nephron architecture and
reestablish normal urine formation (7, 10, 12). The results of the present experiments demonstrate that renal tubule cell repair and regeneration, as reflected by incorporation of radiolabeled thymidine within the kidney, begins as early as 24 h and accelerates between 24 to 48 h after HgClz administration. As demonstrated by histoautoradiography, this renal thymidine incorporation into DNA was essentially confined to tubule cells. This time course of renal thymidine incorporation after HgClz is similar to the initial report that carefully evaluated the time course of renal tubule epithelial cell regeneration after HgClz-induced toxic acute renal failure in rats (7). This report demonstrated that this toxic process resulted in necrosis of proximal tubule cells
FIG. 5. Representative light micrograph of inner cortex (X90) from rat 24 h after HgCl, administration and EGF treatment. Extensive tubule necrosis is also present and similar to that observed in non-EGF-treated animal (compare with Fig. 4).
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 31, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
F442
EGF
AND
TOXIC
ACUTE
during the initial 24 h after HgCl, administration. Epithelial cell regeneration and repair, as measured by radiolabeled thymidine incorporation, began to rise between 24 and 48 h and peaked at 72 h after toxin exposure. This regenerative process resulted in complete relining of the nephron by 4-5 days following HgC12 administration. This process of regeneration and repair by surviving renal tubule cells is most likely dependent on the local production and release of growth-promoting substances (20). EGF may be instrumental in this renal regeneration process after acute tubular necrosis for several reasons. Proximal tubule cells possess EGF receptors (9,16). EGF and TGF-cu, which activate cells via the EGF receptor (8), are the most effective growth-promoting substances identified so far for renal proximal tubule cells (16, 21). Recent evidence has also demonstrated that the cells in the thick ascending limb of Henle and distal convoluted tubule (2, 17) are major sites of synthesis for the precursor of EGF and pre-pro-EGF. The presence of the EGF precursor in these nephron segments may suggest an autocrine pathway for EGF release to promote tubule cell replication after toxic injury. A paracrine pathway for EGF or TGF-cu release may also be initiated after renal tubule cell injury to promote proximal tubule cell regeneration. Platelets and macrophages have been demonstrated to be rich sources of TGF-cu (1, 18), a functional homologue of EGF. Platelet activation and macrophage recruitment after nephrotoxic injury may release TGF-cu locally to promote proximal tubule cell replication. The data presented in these studies demonstrate that exogenously administered EGF can play an important role in the repair and recovery from renal injury after a substantial nephrotoxic event. Exogenous EGF produced an accelerated renal tubule epithelial cell regenerative and repair response compared with normal response times after toxic renal tubule cell injury. This enhanced replicative and repair process led to a shortened time to recover renal excretory function compared with time required in nontreated animals. This improvement in renal excretory function with exogenous EGF administration has also been recently shown for postischemic acute renal failure (10). This EGF-promoted accleration in renal recovery is not likely due to changes in the degree of renal tubule cell injury. Previous work has demonstrated that peak renal tubule cell necrosis occurs within 24 h after HgCl, exposure (13). Histological grading at that time in the groups receiving HgCl, with or without EGF administration in this study demonstrated similar degrees of tubule injury between the two groups. This similarity in the degree of tubule injury is also supported by the fact that the elevations in BUN and serum creatinine values at 24 and 48 h in both groups of HgC12-injected animals either treated with or without EGF were nearly identical, thereby also suggesting similar degrees of cell injury. In fact, the differences in BUN and serum creatinine levels in these two groups only became apparent at 72 h after toxin administration, at a time when renal tubule cell replication and recovery were accelerating. These data support a predominant effect of exogenous EGF to pro-
RENAL
FAILURE
mote repair processes rather than to minimize injurious events. EGF has also been demonstrated to be important in repair processes in other tissues. EGF found in saliva has been suggested to accelerate the repair of cutaneous wounds in rodents as they lick the area of injury (15). Topical administration of EGF has enhanced the repair of skin injuries (4) and cornea1 abrasions (3). The functional EGF analogue, TGF-cu, has also been shown to accelerate regeneration of the epidermis after partial thickness burns or split-thickness incisions in vivo (19) and has recently been demonstrated to be an autocrine pathway for liver regeneration after partial hepatectomy (14). This report demonstrates that EGF also accelerates the repair process of a visceral organ after a significant toxic insult and may eventually have a proven value in clinical circumstances (5). The excellent technical assistance of Ralph Reiss and Kay Brabec, the helpful biostatistical consultation of Dr. Morton Brown, and the expert secretarial support of Sandra Mariott and Cecile Bagrow are appreciated. These studies were supported by the Veterans Administration Research Service and by National Institute of Diabetes and Digestive and Kidney Diseases Grants ROl DK-30819 and PO1 DK-39155. Address for reprint requests: H. D. Humes, Medical Service (Ill), VA Medical Center, 2215 Fuller Rd., Ann Arbor, MI 48105. Received
7 September
1989; accepted
in final
form
7 May
1990.
REFERENCES 1. ASSOIAN, R. K., G. R. GROTENDORST, D. M. MILLER, AND M. B. SPORN. Cellular transformation by coordinated action of three peptide growth factors from human platelets. Nature Lond. 309: 804-806, 1984. 2. BELL, G. I., N. M. FOND, M. M. STEMPIEN, M. A. WORMSTED, D. CAPUT, L. Ku, M. S. URDEA, L. B. RAL, AND R. SANCHEZPESCADOR. Human epidermal growth factor precursor: cDNA sequence, expression in vitro and gene organization. NucZeic Acids Res. 14: 8427-8446,1986. 3. BRIGHTWELL, J. R., S. L. RIDDLE, R. A. EIFERMAN, P. VALENZUELA, P. J. BARR, J. P. MERRYWEATHER, AND G. S. SCHULTZ. Biosynthetic human EGF accelerates healing of Neodecadrontreated primate corneas. Inuest. Ophthalmol. Visual Sci. 26: 105110,1985. 4. BROWN, G. L., L. CURTSINGER, J. R. BRIGHTWELL, D. M. ACKERMAN, G. L. TOBIN, H. C. POLK, C. GEORGE-NASCIEMENTO, P. VALENZUELA, AND G. S. SCHULTZ. Enhancement of epidermal regeneration by biosynthetic epidermal growth factor. J. Exp. Med. 163: 1319-1324,1986. 5. BROWN, G. L., L. B. NANNEY, J. GRIFFEN, A. B. CRAMER, J. M. YANCEY, L. J. CURTSINGER, L. HOLTZIN, G. S. SCHULTZ, M. J. JURKIEWICZ, AND J. B. LYNCH. Enhancement of wound healing by topical treatment with epidermal growth factor. N. Engl. J. Med. 321: 76-79, 1989. 6. CUPPAGE, F. E., M. CHIGA, AND A. TATE. Cell cycle in the regenerating rat nephron following injury with mercuric chloride. Lab. Inuest. 26: 122-126, 1972. 7. CUPPAGE, F. E., N. CUNNINGHAM, AND A. TATE. Nucleic acid synthesis in the regenerating nephron following injury with mercuric chloride (Abstract). Lab. Inuest. 21: 449, 1969. 8. DERYNCK, R. Transforming growth factor alpha. CelZ 54: 593-595, 1988. 9. HUMES, H. D., D. A. CIESLINSKI, T. COIMBRA, AND L. JOHNSON. Mitogenic response of rabbit renal proximal tubule cells to various growth factors (Abstract). Clin. Res. 37: 492A, 1989. H. D., D. A. CIESLINSKI, T. COIMBRA, J. M. MESSANA, 10. HUMES, AND C. GALVAO. Epidermal growth factor enhances renal tubule cell regeneration and repair and accelerates the recovery of renal function in postischemic acute renal failure. J. CLin. Inuest. 84: 1757-1761,1989.
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 31, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
EGF
AND
TOXIC
ACUTE
11. JACKSON, N. M., C. Hsu, G. E. VISSCHER, M. L. VENKATACHALAM, AND H. D. HUMES. Alterations in renal structure and function in a rat model of cyclosporine nephrotoxicity. J. Pharmacol. Exp. Ther. 242: 749-756, 1987. 12. KREISBERG, J. I., AND M. A. VENKATACHALAM. Morphologic factors in acute renal failure. In: Acute RenaZ Failure, edited by B. M. Brenner and J. M. Lazarus. New York: Churchill-Livingstone, 1988, p. 45-66. 13. MCDOWELL, E. M., R. B. NAGLE, R. C. ZALME, J. S. MCNEIL, W. FLAMENBAUM, AND B. F. TR~UMP. Studies on the pathophysiology of acute renal failure. Virchows Arch. B CeZZ Pathol. 22: 173-196, 1976. 14. MEAD, J. E., AND N. FAUSTO. Transforming growth factor-a may be a physiological regulator of liver regeneration by means of an autocrine mechanism. Proc. NatZ. Acad. Sci. USA 86: 1558-1562, 1989. 15. NIALL, M., G. G. RYAN, AND B. J. O’BRIEN. The effect of epidermal growth factor on wound healing in mice. J. Surg. Res. 33: 164-169, 1982. 16. NORMAN, J., B. BADIE-DEZFOOLY, E. P. NORD, I. KURTZ, J. SCHLOSSER, A. CHAUDHARI, AND L. G. FINE. EGF-induced mitogenesis in proximal tubular cells: potentiation by angiotensin II.
RENAL
17.
18.
19.
20.
21.
22.
FAILURE
F443
Am. J. Physiol. 253 (Renal Fluid Electrolyte Physiol. 22): F299F309,1987. RALL, L. B., J. SCOTT, AND G. I. BELL. Mouse prepro-epidermal growth factor synthesis by the kidney and other tissues. Nature Land. 313: 228-231, 1985. RAPPOLEE, D. A., D. MARK, M. BANDA, AND Z. WERB. Wound macrophages express TGF-cu and other growth factors in vivo: analysis by mRNA phenotyping. Science Wash. DC 241: 708-712, 1988. SCHULTZ, G. S., M. WHITE, R. MITCHELL, G. BROWN, J. LYNCH, D. R. TWARDZIK, AND G. J. TODARO. Epithelial wound healing enhanced by transforming growth factor-a an 1 vaccinia growth factor. Science Wash. DC 235: 350-452, 1987. SPORN, M. B., AND A. B. ROBERTS. Peptide growth factors and inflammation, tissue repair, and cancer. J. CZin. Inuest. 78: 329332,1986. STANTON, R. C., AND J. L. SEIFTER. Epidermal growth factor rapidly activates the hexose monophosphate shunt in kidney cells. Am. J. Physiol. 253 (Cell Physiol. 22): C267-C271, 1988. WEINBERG, J. M., P. G. HARDING, AND H. D. HUMES. Alterations in renal cortex cation homeostasis during mercuric chloride and gentamicin nephrotoxicity. Exp. Mol. PathoZ. 39: 43-60, 1983.
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 31, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
M., DEBORAH A. CIESLINSKI, AND H. growth factor accelerates renal repair in mercuric chloride nephrotoxicity. Am. J. Physiol. 259 (Renal Fluid Electrolyte Physiol. 28): F438-F443, 1990.-Repair and recovery of ischemic or nephrotoxic acute renal failure (ARF) are dependent upon renal tubule cell regeneration. Because epidermal growth factor (EGF) is a potent growth promoter to renal tubule cells, experiments were undertaken to assess the effects of exogenous administration of EGF during the recovery phase of HgClz-induced ARF. Rats were administered HgCl, (5 mg/kg SC), and [“Hlthymidine incorporation into renal tissue and blood urea nitrogen (BUN) and serum creatinine concentrations were measured at various times after toxin administration. EGF (20 pg) was administered subcutaneously 2 or 4 h after HgCl, injection. Exogenous EGF resulted in greater levels of renal [‘Hlthymidine incorporation into renal proximal tubule cells compared with those observed in nontreated animals at several time points in the first 48 h after toxic injury. Morphometric analysis of histoautoradiograph sections of renal tissue demonstrated that >96% of labeled cells were tubular in all examined sections. This EGF-related acceleration in DNA synthesis was associated with significantly lower peak BUN and serum creatinine levels, averaging 213 t 23 and 6.54 t 0.72 (SE) mg/dl, respectively, at 3 days in EGF-treated nephrotoxic rats compared with peak levels of 359 t 40 and 9.92 t 1.67 mg/ dl (P < 0.001, n = 7-16) at 5 days in non-EGF-treated nephrotoxic rats. EGF treatment also was associated with a return to near normal BUN and serum creatinine levels approximately 4 days earlier than that observed in non-EGF-treated animals. These findings demonstrate that exogenous EGF accelerates the repair process of the kidney after a severe toxic insult. COIMBRA, TEREZILA DAVID HUMES. Epidermal
acute renal failure; tubule cell regeneration; renal recovery; deoxyribonucleic acid synthesis; thymidine
function
AND H. DAVID Medical Center,
HUMES
Epithelial cells at both ends of the necrotic segments of proximal tubules, as well as other surviving epithelial cells along the denuded surface of the tubular basement membrane, appeared to be responsible for the regenerating cells. In this manner most tubules were relined with new epithelium by day 5. More recent work has demonstrated a time course similar to that observed with HgC1, for renal tubule cell regeneration and renal function recovery after bilateral renal ischemia in the rat (10). These observations suggest that local factors play a significant role in mediating the regeneration process leading to recovery from acute renal failure. This process of regeneration and repair by surviving epithelial cells at the edge of the injury segment is likely dependent on either autocrine or paracrine production of growth-promoting factors. Several growth factors, including platelet-derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor (TGF)-~tl and TGF-P have all been shown to be important in wound healing and reparative processes (20). Of these growth factors, EGF is one of the most potent mitogenic stimuli of renal proximal tubule cells (9, 16). In this regard, a recent report has documented that exogenous administration of EGF early in the recovery phase of ischemic acute renal failure accelerated epithelial cell replication and condensed the time required for renal function recovery (10). This series of studies was therefore carried out to determine whether exogenous EGF has a similar effect of accelerating renal tubule cell regeneration and renal repair in a nephrotoxic model of acute renal failure induced with HgCl,. METHODS
or toxic acute renal failure is a reversible form of organ failure. It has both an injury and recovery phase. The injury phase is due to structural damage to renal epithelial cells, predominantly proximal tubule cells (12). The recovery phase is therefore dependent on the repair and replacement of the injured and necrotic tubule epithelial cells. A classic report carefully evaluated renal epithelial cell regeneration after mercuric chloride (HgCl&-induced toxic acute renal failure in the rat (7). This toxic event resulted in necrosis of proximal tubule cells during the first day after HgCl,. Regeneration of epithelial cells, as reflected by renal thymidine incorporation and radioautography, began by day 2 with immature epithelial cells relining the necrotic areas by day 3. ISCHEMIC
F438
0363-6127/90 $1.50 Copyright
0
Male Sprague-Dawley rats (250-325 g) were administered HgCl, (5 mg/kg SC) followed at 2 or 4 h with a single subcutaneous injection of EGF (20 pg) or sham vehicle (0.9% NaCl). At time intervals of 24, 30, 48, and 72 h after toxin administration, [3H] thymidine incorporation into renal cortex, as well as blood urea nitrogen (BUN) and serum creatinine levels, were determined with methods detailed previously for this laboratory (10, 11). To obtain a complete time course for BUN and serum creatinine levels, some animals were allowed to recover for as long as 12 days after HgClz administration, and blood samples were obtained daily for BUN and serum creatinine levels. To localize and identify the cells incorporating the
1990 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 31, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
EGF
AND
TOXIC
ACUTE
radiolabeled thymidine within the kidney, histoautoradiography of kidney samples was also accomplished by use of methods previously published by this laboratory (10,ll ). Sections were examined at x 176 magnification, and 20-30 fields were counted in renal cortex from an individual animal. The cell type of the labeled cells was identified as either tubular or interstitial. To determine the number of tubule cells incorporating [3H]thymidine after various treatment regimens, 10 random fields in renal cortex and outer medu lla were selected from histoautoradiograph s of kidney specimens, and labeled tubule cells were counted in each field. To ensure that EGF administration did not affect toxin delivery to the kidney and therefore the degree of tubule cell injury, quantitative assessment of morphological alterations induced by HgCl, was accomplished with light microscopy of hematoxylinand eosin-stained sections. By use of a blinded quantitative grading scale previously published by this laboratory (22), the amount of injury in 20 proximal tubules in the inner cortex and 20 proximal tubules in the outer stripe of the outer medulla were graded from 0 ( completely normal tubule) to 5 (maximal loss of tubular architecture with extensive loss of tubular cells). After centering either inner cortex or outer stripe in a low-power field, the high-power objective was positioned to view a field at random. Each field usually contained four or five proximal tubules. If the field had more than five proximal tubules, then the five tubules most central in the field were graded. All reagents used were of the highest grade commercially available. All organic reagents were obtained from Sigma Chemical (St. Louis, MO) unless otherwise indicated. [MethyL-3H] thymidine was obtained from New England Nuclear (Boston, MA). EGF was obtained from AmGen Biologicals (Thousand Oaks, CA). Statistical analysis was performed by use of either Student’s t test or analysis of variance (ANOVA) to identify significant differences between treatment groups. If significant differences between treatment groups were identified by ANOVA, specific comparisons between individual treatment means were accomplished using Tukey’s multiple comparison procedure in conjunction with the Studentized range critical values table. RESULTS
The study design produced a model of reversible nephrotoxic acute renal failure, as demonstrated in Fig. 1. Renal excretory function declined quickly following HgCl, injection, as reflected by BUN levels rising in the first 24 h from an average (&SE) control level of 21 t 2 to a mean of 96 t 4 mg/dl (n = 32). BUN levels peaked to levels of 359 t 40 mg/dl (n = 7) at day 5 after toxic insult followed by progressive improvement thereafter to near normal levels by day 12. As an indication of increases in cell regeneration, [3H]thymidine incorporation into DNA of the kidney began to rise as early as 24 h with dramatic increases between 24 and 48 h after toxic injury (Table 1). Histoautoradiographic studies were performed to determine the type of cells labeled with [3H] thymidine after HgCl, exposure (Fig. 2). Kidney sections from rats at 48 or 72
RENAL
F439
FAILURE
HgCI, -___+ + _---EGF
-
E
:? : I :: * :, : :+..., *** ‘* T------,.-* ** * * * --a--,-, * --~------)-----~------
200
Z 3
m 100
0' 0
12
3
4
5 Time
6 7 (Days)
8
1
9101112
FIG. 1. Time course for blood urea nitrogen (BUN) values in rats administered HgClz at day 0. The group of animals receiving a single dose of epidermal growth factor (EGF; 20 pg) within 2-4 h of HgCl, injection had BUN levels significantly lower at various time points than the animals administered HgCl, not treated with EGF (* P < 0.05, ** P c 0.01, *** P < 0.001, 72 = 5-33).
h after toxic administration were used for histoautoradiographic analysis. Labeling was confined to the nuclei. On average, 97.9% of the labeled cells were tubule cells, and 2.1% of the labeled cells were interstitial cells in the 60 examined fields. In control rats undergoing sham injection, 98.4% of labeled cells were tubule, and 1.6% were interstitial in 90 examined fields. A single dose of exogenously administered EGF at 2 or 4 h after HgCIZ resulted in higher rates of renal thymidine incorporation compared with values observed in non-EGF-treated animals receiving the nephrotoxin at 24, 30, 48, and 72 h (Table 1). When the EGF treatment group was compared with the nontreated group, the effect of EGF to increase renal thymidine incorporation was highly significant by ANOVA (P = 0.0011). Thus EGF significantly enhanced renal thymidine incorporation during the period of peak replicative and repair response of renal tubule cells to HgClz-induced cell necrosis. Of note, EGF treatment of control rats not receiving HgC12 did not demonstrate any increase in [3H]thymidine incorporation in kidneys. At 24 h and after a single dose of EGF in rats undergoing sham HgClz injection, renal [3H]thymidine incorporation averaged 13 t 4 x lo3 disintegrations per minute (dpm)/mg DNA, compared with a value of 12 t 2 X lo3 dpm/mg DNA in nonEGF-treated sham animals (P = NS, n = 4). Histoautoradiographic studies were performed to determine the location of the cells labeled with [3H]thymidine in kidney sections from rats treated with EGF at 48 h after HgCl, injection. Labeling was again confined to nuclei. On average, 96.3% of labeled cells were identified as tubule, and 3.7% labeled cells were identified as interstitial cells in 60 fields. To test the effect of EGF on renal tubule cell replication after HgClz-induced injury in a more absolute manner than measurement of the specific activity of renal [3H] thymidine (Table l), the number of labeled cells in renal cortex and outer medulla was determined at 24 and 48 h after HgC12 administration in both treatment groups. At 24 h EGF treatment increased the number of tubule cells incorporating [3H] thymidine, as assessed by
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 31, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
F440 TABLE
EGF AND TOXIC
ACUTE
RENAL
FAILURE
1. Effect of EGF on renal thymidine incorporation after HgC12 [3H]Thymidine Incorporation, dpm Oh
24 h
30
x
104/mg DNA
h
48
h
12
h
0.13rto.04 HgCl, 8.42+1.08 20.37k4.24 64.16k7.59 99.80+28.04 HgClz + EGF 0.12&0.03 13.81&1.28* 42.03k5.45t 80.52k19.52 116.24+13.80 Values are means + SE; n = 4-8 animals for each group. Time course for renal thymidine incorporation in rats administered 5 mg/kg HgC12. Epidermal growth factor (EGF) was administered 2-4 h after nephrotoxin administration. Relative to non-EGF treatment group, * P < 0.02 and t P < 0.005 by one-way ANOVA and Tukey’s multiple range procedure.
FIG. 2. Histoautoradiography of renal cortex of a kidney from a rat 72 h after HgCl, injection. [3H]thymidine label is observed as black-silver grains above tubule cell nuclei (magnification x500).
histoautoradiography, from 1.6 f 0.4 in the nontreated groups to 7.5 + 1.8 labeled cells/field in the treated group (P < 0.02, n = 3 rats and 60 fields for each group). Similarly at 48 h, EGF increased labeled tubule cells from a nontreated value of 12.1 & 1.8 to 20.9 + 2.1 cells/ field (P < 0.01, n = 3 rats and 60 fields for each group). Thus, EGF significantly enhanced renal tubule cell replication in the early reparative phase after nephrotoxic injury as assessedboth by specific activity of [3H]thymidine in the kidney and by absolute numbers of renal tubule cells incorporating labeled thymidine into DNA. Associated with this EGF-induced enhancement of tubule cell thymidine incorporation, the time course of renal excretory failure after nephrotoxic injury, as measured by both BUN and serum creatinine levels, was dramatically altered by EGF treatment as demonstrated in Figs. 1 and 3. EGF-treated animals had significantly lower peak BUN and serum creatinine levels, averaging 213 rt 23 and 6.54 + 0.72 mg/dl, respectively, at 3 days after HgClz injection, compared with peak levels of 359 + 40 and 9.92 -I- 1.67 mg/dl (P < 0.001, n = 7-16) at 5 days in nontreated nephrotoxic rats. EGF treatment also was associated with a return to near normal BUN and serum creatinine levels in EGF-treated animals on day 8,4 days earlier than that observed in non-EGF-treated animals. EGF treatment did not alter the degree of Hg2+ presented to the kidney, because no difference in histological
HgC’z
I2 r
HgClp. . . +. . EGF
-
0
2
4
5
Time
8
10
(days)
FIG. 3. Time course for serum creatinine values for animals with HgClz-induced acute renal failure. Animals treated with epidermal growth factor (EGF) had significantly lower (* P < 0.05, ** P < 0.01, n = 4-16) values compared with untreated rats with toxic renal injury.
score of tubule cell injury was demonstrated between the nephrotoxic rats treated or not treated with EGF. Histological grading was assessedat 24 h, a time at which the peak of HgCl,-induced toxic renal injury occurs and just before the accelerated regeneration repair phase (6, 7, 13). At 24 h, the mean histological scores in the inner cortex and outer stripe were 2.92 + 0.13 and 4.70 + 0.07, respectively, in the nephrotoxic non-EGF-treated rats vs. scores of 3.25 rt 0.18 and 4.65 & 0.08, respectively, in the nephrotoxic EGF-treated rats (n = 120 fields from 3
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 31, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
EGF AND TOXIC
ACUTE
RENAL
FAILURE
F441
FIG. 4. Representative light micrograph of inner cortex (x90) from rat 24 h after HgCl, administration. Extensive tubule necrosis is present with necrotic debris filling tubular lumina.
animals in each treatment group, P = NS for both scores). As depicted in Figs. 4 and 5, at 24 h after HgCL, extensive necrosis was present in proximal tubule segments with no difference in the degree of tubule cell necrosis observed histologically between the EGFtreated and nontreated groups receiving HgCL DISCUSSION
Because acute renal excretory failure from nephrotoxic or ischemic stress is due to segmental tubule cell necrosis, the reversibility of nephrotoxic or ischemic acute renal failure is dependent upon renal epithelial cell regeneration to reconstruct normal nephron architecture and
reestablish normal urine formation (7, 10, 12). The results of the present experiments demonstrate that renal tubule cell repair and regeneration, as reflected by incorporation of radiolabeled thymidine within the kidney, begins as early as 24 h and accelerates between 24 to 48 h after HgClz administration. As demonstrated by histoautoradiography, this renal thymidine incorporation into DNA was essentially confined to tubule cells. This time course of renal thymidine incorporation after HgClz is similar to the initial report that carefully evaluated the time course of renal tubule epithelial cell regeneration after HgClz-induced toxic acute renal failure in rats (7). This report demonstrated that this toxic process resulted in necrosis of proximal tubule cells
FIG. 5. Representative light micrograph of inner cortex (X90) from rat 24 h after HgCl, administration and EGF treatment. Extensive tubule necrosis is also present and similar to that observed in non-EGF-treated animal (compare with Fig. 4).
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 31, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
F442
EGF
AND
TOXIC
ACUTE
during the initial 24 h after HgCl, administration. Epithelial cell regeneration and repair, as measured by radiolabeled thymidine incorporation, began to rise between 24 and 48 h and peaked at 72 h after toxin exposure. This regenerative process resulted in complete relining of the nephron by 4-5 days following HgC12 administration. This process of regeneration and repair by surviving renal tubule cells is most likely dependent on the local production and release of growth-promoting substances (20). EGF may be instrumental in this renal regeneration process after acute tubular necrosis for several reasons. Proximal tubule cells possess EGF receptors (9,16). EGF and TGF-cu, which activate cells via the EGF receptor (8), are the most effective growth-promoting substances identified so far for renal proximal tubule cells (16, 21). Recent evidence has also demonstrated that the cells in the thick ascending limb of Henle and distal convoluted tubule (2, 17) are major sites of synthesis for the precursor of EGF and pre-pro-EGF. The presence of the EGF precursor in these nephron segments may suggest an autocrine pathway for EGF release to promote tubule cell replication after toxic injury. A paracrine pathway for EGF or TGF-cu release may also be initiated after renal tubule cell injury to promote proximal tubule cell regeneration. Platelets and macrophages have been demonstrated to be rich sources of TGF-cu (1, 18), a functional homologue of EGF. Platelet activation and macrophage recruitment after nephrotoxic injury may release TGF-cu locally to promote proximal tubule cell replication. The data presented in these studies demonstrate that exogenously administered EGF can play an important role in the repair and recovery from renal injury after a substantial nephrotoxic event. Exogenous EGF produced an accelerated renal tubule epithelial cell regenerative and repair response compared with normal response times after toxic renal tubule cell injury. This enhanced replicative and repair process led to a shortened time to recover renal excretory function compared with time required in nontreated animals. This improvement in renal excretory function with exogenous EGF administration has also been recently shown for postischemic acute renal failure (10). This EGF-promoted accleration in renal recovery is not likely due to changes in the degree of renal tubule cell injury. Previous work has demonstrated that peak renal tubule cell necrosis occurs within 24 h after HgCl, exposure (13). Histological grading at that time in the groups receiving HgCl, with or without EGF administration in this study demonstrated similar degrees of tubule injury between the two groups. This similarity in the degree of tubule injury is also supported by the fact that the elevations in BUN and serum creatinine values at 24 and 48 h in both groups of HgC12-injected animals either treated with or without EGF were nearly identical, thereby also suggesting similar degrees of cell injury. In fact, the differences in BUN and serum creatinine levels in these two groups only became apparent at 72 h after toxin administration, at a time when renal tubule cell replication and recovery were accelerating. These data support a predominant effect of exogenous EGF to pro-
RENAL
FAILURE
mote repair processes rather than to minimize injurious events. EGF has also been demonstrated to be important in repair processes in other tissues. EGF found in saliva has been suggested to accelerate the repair of cutaneous wounds in rodents as they lick the area of injury (15). Topical administration of EGF has enhanced the repair of skin injuries (4) and cornea1 abrasions (3). The functional EGF analogue, TGF-cu, has also been shown to accelerate regeneration of the epidermis after partial thickness burns or split-thickness incisions in vivo (19) and has recently been demonstrated to be an autocrine pathway for liver regeneration after partial hepatectomy (14). This report demonstrates that EGF also accelerates the repair process of a visceral organ after a significant toxic insult and may eventually have a proven value in clinical circumstances (5). The excellent technical assistance of Ralph Reiss and Kay Brabec, the helpful biostatistical consultation of Dr. Morton Brown, and the expert secretarial support of Sandra Mariott and Cecile Bagrow are appreciated. These studies were supported by the Veterans Administration Research Service and by National Institute of Diabetes and Digestive and Kidney Diseases Grants ROl DK-30819 and PO1 DK-39155. Address for reprint requests: H. D. Humes, Medical Service (Ill), VA Medical Center, 2215 Fuller Rd., Ann Arbor, MI 48105. Received
7 September
1989; accepted
in final
form
7 May
1990.
REFERENCES 1. ASSOIAN, R. K., G. R. GROTENDORST, D. M. MILLER, AND M. B. SPORN. Cellular transformation by coordinated action of three peptide growth factors from human platelets. Nature Lond. 309: 804-806, 1984. 2. BELL, G. I., N. M. FOND, M. M. STEMPIEN, M. A. WORMSTED, D. CAPUT, L. Ku, M. S. URDEA, L. B. RAL, AND R. SANCHEZPESCADOR. Human epidermal growth factor precursor: cDNA sequence, expression in vitro and gene organization. NucZeic Acids Res. 14: 8427-8446,1986. 3. BRIGHTWELL, J. R., S. L. RIDDLE, R. A. EIFERMAN, P. VALENZUELA, P. J. BARR, J. P. MERRYWEATHER, AND G. S. SCHULTZ. Biosynthetic human EGF accelerates healing of Neodecadrontreated primate corneas. Inuest. Ophthalmol. Visual Sci. 26: 105110,1985. 4. BROWN, G. L., L. CURTSINGER, J. R. BRIGHTWELL, D. M. ACKERMAN, G. L. TOBIN, H. C. POLK, C. GEORGE-NASCIEMENTO, P. VALENZUELA, AND G. S. SCHULTZ. Enhancement of epidermal regeneration by biosynthetic epidermal growth factor. J. Exp. Med. 163: 1319-1324,1986. 5. BROWN, G. L., L. B. NANNEY, J. GRIFFEN, A. B. CRAMER, J. M. YANCEY, L. J. CURTSINGER, L. HOLTZIN, G. S. SCHULTZ, M. J. JURKIEWICZ, AND J. B. LYNCH. Enhancement of wound healing by topical treatment with epidermal growth factor. N. Engl. J. Med. 321: 76-79, 1989. 6. CUPPAGE, F. E., M. CHIGA, AND A. TATE. Cell cycle in the regenerating rat nephron following injury with mercuric chloride. Lab. Inuest. 26: 122-126, 1972. 7. CUPPAGE, F. E., N. CUNNINGHAM, AND A. TATE. Nucleic acid synthesis in the regenerating nephron following injury with mercuric chloride (Abstract). Lab. Inuest. 21: 449, 1969. 8. DERYNCK, R. Transforming growth factor alpha. CelZ 54: 593-595, 1988. 9. HUMES, H. D., D. A. CIESLINSKI, T. COIMBRA, AND L. JOHNSON. Mitogenic response of rabbit renal proximal tubule cells to various growth factors (Abstract). Clin. Res. 37: 492A, 1989. H. D., D. A. CIESLINSKI, T. COIMBRA, J. M. MESSANA, 10. HUMES, AND C. GALVAO. Epidermal growth factor enhances renal tubule cell regeneration and repair and accelerates the recovery of renal function in postischemic acute renal failure. J. CLin. Inuest. 84: 1757-1761,1989.
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 31, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
EGF
AND
TOXIC
ACUTE
11. JACKSON, N. M., C. Hsu, G. E. VISSCHER, M. L. VENKATACHALAM, AND H. D. HUMES. Alterations in renal structure and function in a rat model of cyclosporine nephrotoxicity. J. Pharmacol. Exp. Ther. 242: 749-756, 1987. 12. KREISBERG, J. I., AND M. A. VENKATACHALAM. Morphologic factors in acute renal failure. In: Acute RenaZ Failure, edited by B. M. Brenner and J. M. Lazarus. New York: Churchill-Livingstone, 1988, p. 45-66. 13. MCDOWELL, E. M., R. B. NAGLE, R. C. ZALME, J. S. MCNEIL, W. FLAMENBAUM, AND B. F. TR~UMP. Studies on the pathophysiology of acute renal failure. Virchows Arch. B CeZZ Pathol. 22: 173-196, 1976. 14. MEAD, J. E., AND N. FAUSTO. Transforming growth factor-a may be a physiological regulator of liver regeneration by means of an autocrine mechanism. Proc. NatZ. Acad. Sci. USA 86: 1558-1562, 1989. 15. NIALL, M., G. G. RYAN, AND B. J. O’BRIEN. The effect of epidermal growth factor on wound healing in mice. J. Surg. Res. 33: 164-169, 1982. 16. NORMAN, J., B. BADIE-DEZFOOLY, E. P. NORD, I. KURTZ, J. SCHLOSSER, A. CHAUDHARI, AND L. G. FINE. EGF-induced mitogenesis in proximal tubular cells: potentiation by angiotensin II.
RENAL
17.
18.
19.
20.
21.
22.
FAILURE
F443
Am. J. Physiol. 253 (Renal Fluid Electrolyte Physiol. 22): F299F309,1987. RALL, L. B., J. SCOTT, AND G. I. BELL. Mouse prepro-epidermal growth factor synthesis by the kidney and other tissues. Nature Land. 313: 228-231, 1985. RAPPOLEE, D. A., D. MARK, M. BANDA, AND Z. WERB. Wound macrophages express TGF-cu and other growth factors in vivo: analysis by mRNA phenotyping. Science Wash. DC 241: 708-712, 1988. SCHULTZ, G. S., M. WHITE, R. MITCHELL, G. BROWN, J. LYNCH, D. R. TWARDZIK, AND G. J. TODARO. Epithelial wound healing enhanced by transforming growth factor-a an 1 vaccinia growth factor. Science Wash. DC 235: 350-452, 1987. SPORN, M. B., AND A. B. ROBERTS. Peptide growth factors and inflammation, tissue repair, and cancer. J. CZin. Inuest. 78: 329332,1986. STANTON, R. C., AND J. L. SEIFTER. Epidermal growth factor rapidly activates the hexose monophosphate shunt in kidney cells. Am. J. Physiol. 253 (Cell Physiol. 22): C267-C271, 1988. WEINBERG, J. M., P. G. HARDING, AND H. D. HUMES. Alterations in renal cortex cation homeostasis during mercuric chloride and gentamicin nephrotoxicity. Exp. Mol. PathoZ. 39: 43-60, 1983.
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 31, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
