Epidermal growth factor suppresses renal tubular apoptosis following ureteral obstruction

BASIC SCIENCE CME ARTICLE

ELSEVIER

EPIDERMAL GROWTH FACTOR SUPPRESSES RENAL TUBULAR APOPTOSIS FOLLOWING URETERAL OBSTRUCTION WILLIAM A. KENNEDY II, RALPH BUTTYAN, EDUARDO GARCIA-MONTES, VIVETTE D'AGATI, CARL A. OLSSON, AND IHOR S. SAWCZUK

ABSTRACT Objectives. Acute unilateral ureteral obstruction (UUO) results in ipsilateral hydronephrosis characterized by a decrease in epidermal growth factor (EGF) mRNA expression and EGF protein levels in the distal renal tubules. UUO results in programmed cell death with increases in the characteristic markers of apoptosis. To suppress the apoptotic response during UUO, recombinant EGF was administered during renal obstruction and the ensuing molecular and histologic changes were studied. Methods. Mature Sprague-Dawley rats underwent left ureteral obstruction and the kidneys were harvested at 24, 48, and 72 hours. Markers of apoptosis included DNA laddering pattern on agarose gel electrophoresis, in situ gap labeling of fragmented DNA for quantitative apoptotic body determination, polyadenylated mRNA expression of SGP-2, and in situ hybridization for sulfated glycoprotein-2 (SGP-2) mRNA. Studies were repeated in rats following administration of 10, 20, and 40 #g of subcutaneous recombinant EGF on a daily basis after UUO. Results. Subcutaneous injection of EGF into unilaterally obstructed rats promotes renal tubular epithelial cell regeneration, as demonstrated by increased cortical mitotic activity. Systemic EGF supplementation in these unilaterally obstructed rats also resulted in a decrease in the intensity of the DNA laddering pattern associated with renal tubular apoptosis. An in situ labeling procedure to identify apoptotic nuclei in the ureterally obstructed kidneys revealed a 50% reduction in apoptosis after EGF administration. Northern blot analysis and in situ hybridization for SGP-2 mRNA or clusterin gene product also revealed a decreased expression in the obstructed and EGF-treated renal parenchyma. Conclusions. These data suggest that EGF, apart from its known role as a mitogenic substance for renal tubular epithelial cells, is also a critical in vivo renal cell survival factor for the developmentally mature kidney. UROLOGY49: 973-980, 1997. © 1997, Elsevier Science Inc. All rights reserved.

rydronephrosis, resulting from the impaired

.flow of urine as a consequence of structural H or functional abnormalities of the urinary tract, is

From the Departments of Urology and Pathology, College of Physicians and Surgeons, Columbia University, New Yorh, New York; and Department of Urology, Maimonides Medical Center, Brooklyn, New York Supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases U.S.S. and R.B.). W.A.K was the recipient of an American Foundation for Urological Diseases/ National Kidney Foundation Research Fellowship Award. Reprint requests: William A. Kenned), II, M.D., Department of Urology, S-287, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305-5118 Submitted: June 2, 1996, accepted (with revisions): January 8, 1997 © 1 9 9 7 , ELSL-\.'IER SCIEN(E INC. ,,\1 L RIGHTS RES[:.R\ED

a common clinical entity. The National Kidney and Urological Diseases Advisory Board reported in 1990 that approximately 397,000 hospital admissions per year were a direct result of obstructive uropathy. Hydronephrosis is a manifestation of a variety of urologic diseases which include obstructing renal and ureteral calculi, ureteral pelvic junction obstruction, congenital ureteral anomalies (ie, valves, obstructive megaureters, and ureteroceles), benign prostatic hyperplasia, posterior urethral valves, and dysfunctional voiding. Hydronephrosis is a degenerative condition that can be experimentally induced by surgical ligation of the ureter. ~ Histopathologic, biochemical, and genetic studies have demonstrated that the renal atrophy associated with this condition is related to 0090-4295197t$17.00 PlI 5 0 0 9 0 - 4 2 9 5 ( 9 7 ) 0 0 1 0 1 - 5

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the onset of p r o g r a m m e d cell death or apoptosis, primarily in the distal tubular e p i t h e l i u m } In contrast to necrosis, the m o r e classically u n d e r s t o o d m o d e of cell death, apoptosis is a genetically active cell death, 3 sometimes requiring the dying cell to synthesize ribonucleic acid (RNA) and proteins for its occurrence. + r This r e q u i r e m e n t for genetic activity is consistent with the ability to identify specific gene p r o d u c t s w h o s e expression is i n d u c e d d u r i n g h y d r o n e p h r o s i s , including the sulfated glycoprotein-2 (SGP-2) or clusterin gene product, certain heat s h o c k proteins (hsp-70), an immediate early transcription factor (c-los), and the c-myc product, otherwise described in association with cell growth, s The expression of these latter p r o d u c t s is k n o w n to be highly sensitive to events that activate cellular signal t r a n s d u c t i o n pathways, especially by g r o w t h factor action. The onset of apoptosis in the o b s t r u c t e d rat k i d n e y is associated with a cessation of the constitutively high expression of epidermal g r o w t h factor (EGF) m R N A as well as EGF protein synthesis in the distal c o n v o l u t e d tubules. 9 ~t W i t h the reversal of renal obstruction, p r e - p r o E G F m R N A levels have been s h o w n to slowly r e t u r n to baseline. H It is well established that certain g r o w t h factor deficiencies initiate apoptosis for m a n y cell types in culture and at naturally o c c u r r i n g develo p m e n t a l sites, hA3 For example, recent studies in developing and degenerative n e u r o n a l systems d e m o n s t r a t e that local application of g r o w t h factors can rescue m o t o r n e u r o n s from p r o g r a m m e d cell death in vivo. 14-16 Studies on d e v e l o p m e n t a l renal systems have also revealed that EGF a d m i n istration can inhibit cell death associated with normal kidney development. ~7'1s W e report here that EGF administered s u b c u t a n e o u s l y to ureterally o b s t r u c t e d rats suppresses apoptosis of epithelial cells in the distal tubules and collecting ducts of the h y d r o n e p h r o t i c k i d n e y and p r o m o t e s tubular epithelial cell regeneration. These data suggest that EGF, aside from its k n o w n role as a mitogenic substance for renal tubular epithelial cells, is also a critical in vivo renal cell survival factor for the developmentally m a t u r e kidney. M A T E R I A L AND M E T H O D S ANIMAL MODEL Adult Sprague-Dawley rats weighing 250 to 300 g were housed under standard conditions of heat and humidity on a 12-hour light cycle with unlimited access to water and a standard diet and were cared for in accordance with institutional guidelines. Using sodium pentobarbital anesthesia (0.04 mg/ kg intraperitoneally), animals underwent left proximal complete ureteral ligation with a 4-0 silk suture. Animals were divided into treatment and no treatment arms. Those enrolled in the treatment arm received subcutaneous EGF (Recombinant Epidermal Growth Factor, R & D Systems, Minneapolis, Minn) supplementation at doses of 10, 20, or 40 ~g, in 1 mL 97/-+

TABLE

I.

M i t o t i c nuclear scores in the

cortical tubular epithelium of hydronephrotic rat kidneys supplemented with epidermal growth factor Dose (/xg EGF/day)

Total No. of Mitotic Figures

MNS* (mean _+ SE) P Value*

0 10 20

15 41 38

0.75 + 0.14 2.00 + 0.42 1.90 + 0.33

0.011 <0.001

40

45

2.25 _+ 0.35

<0.001

K*~: EGF - ephbtmal growth.lactm: /VLN'S Imtoth m~ch'u~ scotc.s * MNS is presented as the mean number o] .moses pcl 20 high powclcd lieh/s. Quantitation was performed on q-lain seuions q] parqOin-cmbeddcd .'hal IJss[it" All ,teidncks anal yzcd '~;crc harvested at 24 horns aflt'l obst~t, tion aml EGF adDlilliSllotiOn. Sign{[ica.tlv mt.e ~ortical tubula~ ctmhelial ~clls tmdclwcnl icplit¢ttion ¢{lh'~ tlcatment with EGF compmcd with obstrl,ltted and untutored kidm:'vs (5,tudent's two-tailed t test).

of phosphate-buffered saline (PBS) each day. EGF supplementation was begun within 15 minutes of surgical ligation of the ureter. The other animals did not receive any EGF supplementation. Animals were killed at 24, 48, and 72 hours postoperatively. Two animals were used for each of the dose/ harvest time subdivisions of the experiment. Sham-operated animals served as controls. The kidneys were rapidly harvested and snap-frozen in liquid nitrogen for molecular biologic analysis and portions were preserved in 4% paraformaldehyde/PBS solution for histologic studies.

LIGHT MICROSCOPIC QUANTITATION OF MITOTIC FIGURES Tissues were fixed in 4% paraformaldehyde/PBS solution and embedded in paraffin. Microsections of 2/lm were stained with hematoxylin-eosin (H&E). The number of mitotic figures from each of 20 randomly selected high-powered light microscopic fields were recorded by a renal pathologist (V.D.) who was blinded to EGF supplementation status of the tissue. Statistical analysis was carried out using a twotailed Student's t test. D N A FRAGMENTATION ANALYSIS DNA was extracted from pulverized frozen tissue using an affinity column chromatography procedure provided with the A.S.A.P. kit of Boehringer Mannheim, Inc. (Indianapolis, Ind). DNA concentrations were ascertained by ultraviolet spectrophotometry at 260 nm, and 5-#g aliquots of DNA were electrophoresed on a 1.5% agarose gel. The gel was stained with ethidium bromide and visualized under uhraviolet transillumination. ~ IN SITU GAP LABELING OF FRAGMENTED NUCLEAR D N A (APOPTOTIC BODY STAIN) In situ gap labeling2° 23 was begun by fixing tissues in 4% paraformaldehyde/PBS solution and embedding them in paraffin. Microsections of 2 #m were rehydrated and then digested with pepsin 0.1%, pH 1.5, at 37°C for 30 minutes and washed in cold water. Nuclear DNA was denatured at 70°C in 2 × saline-sodium citrate (SSC), pH 7.0, for 15 minutes and washed in cold water. Slides were then incubated in the reaction buffer of 50 mM Tris pH 7.5, 5 nM MgC12; 10 mM beta-mercaptoethanol; bovine serum albumin (BSA) 0.005%. DNA polymerization was processed by using the Klenow flaglnent of the DNA polymerase (50 UI/mL), containing digoxUROLOGY 49 (0), 1997

a

b 48 hr,, ze hrs ~

-I- -

EGF (tzg I day) 40 20 10 '0"

n,

"0"

,xn,

q- -

1. Nuclear DNA fragmentation analysis in (a) obstructed~untreated kidneys ( - ) and obstructed/EGF treated (20 t~g/day) kidneys (+) harvested at 48 and 72 hours postoperatively and (b) sham-operated control kidney (NI) and obstructed kidneys harvested at 24, 48, and 72 hours postoperatively after treatment with 10, 20, or 40 l~g/day of EGF. Obstructed and low dose EGF supplemented kidneys (10 #g/day) show the distinctive ladder pattern of apoptotic ceil death consisting of DNA fragments of sizes in multiples of 180 bp. A decrease in DNA fragmentation is observed in the obstructed and high dose EGF supplemented kidneys (40 t~g/day). MW = comparative molecular weight markers. FIGURE

igenin-labeled deoxy-uridine. This reaction was processed at 15°C for 40 minutes. Slides were then washed in the reaction buffer for 5 minutes and incubated for 1 hour at room temperature in a DNA blocking reagent (Boehringer Mannheim) and for 40 minutes with the blocking agent containing an alkaline phosphatase labeled mouse anti-digoxigenin monoclonal antibody at a concentration of 1:500. Slides were then washed in a maleate buffer at pH 7.0 and in alkaline phosphatase buffer (100 nM Tris pH 9.5; 100 mM NaCI; 50 mM MgC12). Alkaline phosphatase activity was detected with a reagent solution containing nitro blue terazolium/5-bromo-4chloro-3-indolylphosphate (NBT/BC1P). Slides were lightly counterstained with 1% light green, air dried, and mounted in resin after a brief wash in xylene. Computerized image analysis was used to obtain objective quantitative counts of the apoptotic nuclei in 10 randomly selected high powered fields in both the renal cortex and renal medulla. This was carried out on a Cell Analysis System 200 Image Analyzer.

ISOLATION OV R N A AND POLYADENYLATED (POLY ( A ) + ) SELECTION Tissue that was snap-frozen was pulverized at -70°C in the presence of liquid nitrogen and then homogenized in 5 M guanidine thiocyanate containing 5% 2-mercaptoethanol.2~ RNA was precipitated selectively with lithium chloride overnight at 4°C. The homogenate was then centrifuged and precipitated with ethanol at -20°C by addition of sodium acetate. Poly(A)+ mRNA was then selected by oligo-dT cellulose chromatography2~ and quantified by ultraviolet spectrophotometry at 260 nm.

R N A GEL ELECTROPHORESISAND NORTHERN BLOTTING Five-microgram aliquots of poly(A)+ mRNA were denatured in 50% formamide and 2.2 M formaldehyde at 65°C for UROLOGY 49 (6), 1997

5 minutes. Electrophoresis was performed in denaturing 1.2% agarose, 2.2 M formaldehyde gels at 70 V for 3 hours. The gel was then soaked in 20 × SSC for 1 hour followed by transfer to nylon filters in 20 × SSC by capillary blotting techniques for 24 hours. The blot was fixed by baking at 80°C under vacumn for 2 to 3 hours. Use of nylon filters over nitrocellulose has permitted higher retention of blotted nucleic acids and allowed for increased number of sequential hybridizations.

NORTHERN HYBRIDIZATION WITH e D N A PROBES The cDNA probe for SGP-2 [clusterin] was labeled with 32p deoxynucleotides by primer extension technique. 26 Typical incorporation procedures yield probes with specific activities of 3 to 8 × 10" disintegrations per minute per microgram DNA. Blots were prehybridized, then hybridized with denatured probes in a solution of 6 × SSC, 0.5% sodium dodecyl sulfate, 5 mM ethylenediaminetetraacetic acid, 5 x Denhardt's solution, 50% formamide, and 200 /2L denatured salmon sperm DNA. Filters were then washed to a final stringency of 0.1 X SSC at 55°C. Autoradiograms were then prepared by exposing the hybridized filters to X-OMAT AR film. Quantitative densitometry was performed on autoradiograms using a computer based measurement of the integral of area and density on a Joyce-Loebel Chromoscan 3 Densitometer.

IN SITU HYBRIDIZATION FOR SGP-2 m R N A In situ hybridization27 was begun by fixing tissues in 4% paraformaldehyde/PBS solution and embedding them in paraffin. RNA probes were prepared from T7/SP6 expression vector [pGEM-vector, Promega] containing a partial eDNA insert for SGP-2 [clusterin] (PG21-04) inserted between the T7 and SP6 RNA polymerase promoter sites. In vitro transcription was performed on a linearized plasmid with either T7 or SP6 polymerase and a nucleotide mixture containing digoxigenin

975

L-

r r ~~

• 7 ~

b

6

of tubular epithelium following ischemic renal injury. is This therapy was proposed based on the knowledge that EGF is a potent mitogen for renal tubular cells in culture. > Light microscopic quantitation of mitotic figures was performed on renal tissue from mature, ureterally obstructed Sprague-Dawley rats supplemented with subcutaneously administered EGF in doses ranging from 10 to 40 #g. Indeed, daily subcutaneous injections of EGF into these ureterally obstructed rats increased the mitotic activity of the tubular epithelial cells. The mitotic nuclear score (MNS) of the renal cortex more than doubled with the administration of EGF to the obstructed kidneys (Table I). These data demonstrate that EGF administration following ipsilateral ureteral obstruction can stimulate proliferation of tubular epithelial cells in viva, and indicate that systemic EGF treatment has the potential to affect degenerative renal conditions initiated by insults other than ischemia. D N A FRAGMENTATION ASSAY SHOWS REDUCTION OF APOPTOSIS WITH EGF SUPPLEMENTATION FOLLOWING HYDRONEPHROSIS

FIGURE 2. In situ gap labeling of fragmented nuclear DNA in (a) sham-operated control cortical kidney, (b) obstructed cortical kidney, and (c) obstructed cortical kidney treated with 20 l~g/day EGF and harvested at 72 hours postoperatively. Dark blue staining nuclei have fragmented nuclear DNA characteristic of apoptosis. Tissues are counterstained with 1% light green. Photomicrographs ore reduced from optical magnification ×200.

labeled deoxy-Uridine (Boehringer Mannheim). Following hybridization, the 2-#m slides were incubated in a solution containing an alkaline phosphatase labeled mouse anti-digoxigenin monoclonal antibody at a concentration of 1:500. Alkaline phosphatase activity was detected with a reagent solution containing NBT/BCIP.

RESULTS

Further effects of EGF administration can be confirmed by an analysis of the molecular markers characteristic of apoptosis. Apoptosis can be identified by a unique pattern of DNA degradation in which the DNA of the intranucleosomal region is preferentially digested. As a result, DNA extracted from such tissues has a characteristic ladder of DNA fragments on agarose gel electrophoresis. 3° Electrophoresis of DNA (Fig. 1) isolated from renal tissue of obstructed and obstructed/EGF supplemented kidneys, harvested at 24, 48, and 72 hours following complete left proximal ureteral ligation, was performed. An intense DNA laddering pattern characteristic of apoptosis is evident in the obstructed and untreated rat kidneys compared with a diminished pattern in the EGF-supplemented kidneys (Fig. la). Suppression of DNA fragmentation is confirmed in a dose-response experiment. Increasing daily doses of EGF resulted in a decrease in the intensity of the DNA laddering pattern over time (Fig. lb). Although the DNA fragmentation pattern is very apparent in the rats treated at the lowest dose of EGF (10 /ag/day), higher doses suppress the laddering pattern. The degree of DNA fragmentation observed in the obstructed kidneys supplemented with 40 #g/day of EGF and harvested at 48 and 72 hours is comparable to that of the sham-operated control kidneys.

ACTIVITY OF TUBULAR EPITHELIAL CELLS

I-IISTOLOGIC EVIDENCE THAT EGF SUPPLEMENTATION DECREASES RENAL TUBULAR CELL APOPTOSIS

Supplemental EGF therapy previously has been advocated as a means of accelerating regeneration

In situ gap labeling (ISGL) of fragmented nuclear DNA allows for selective immunohistochem-

E G F SUPPLEMENTATION INCREASES MITOTIC

976

UROLOGY 49 (6), 19!)7

a

b

Densitometric Units 3,000

+1

2,500-

2,000

2.0 kb 1,500

SGP-2 mRNA

1,000

•

500

18S Ribosomal RNA

0 72 hr

24 hr

TREATED

72 hr

24 hr

NL

UNTREATED

F I G U R E 3. (a) Northern blot analysis for SGP-2 gene expression in sham-operated control kidney (NI), obstructed and untreated kidneys harvested at 24 and 72 hours, and obstructed and EGF-treated kidneys (10 l~g/day) harvested at 24 and 72 hours. 2.0 kilobase (kb) SGP-2 rnRNA transcript is depicted in representative blot. Five micrograms of poly(A)+ rnRNA/lane loading equivalency was confirmed by 18s ribosomal band. (b) Graphic representation of densitometric analysis of SGP-2 mRNA expression.

ical labeling of apoptotic nuclei in tissue. 2°-23 Sections of kidneys from the sham-operated control and obstructed and obstructed/EGF supplemented rats were labeled in this manner. The dark blue stained nuclei identified by this technique were infrequently observed in the sham-operated control rat kidney (Fig. 2a). Conversely, the obstructed rat cortex (Fig. 2b) illustrates a disruption of normal renal architecture with dilatation of collecting and distal tubules in addition to an abundance of tubular epithelial cells staining by this technique. The obstructed/EGF supplemented cortical tissues (Fig. 2c) still possess the tubular dilatation that results from obstruction; however, there is a remarkable reduction in the number of in situ gap labeled nuclei. Similar changes were noted in the rat medullary tissue during obstruction and obstruction/EGF supplementation (data not shown). Computerized image analysis of these tissues with a Cell Analysis System 200 yielded quantitative counts of the apoptotic nuclei based on 10 randomly selected high powered fields in both the renal cortex and renal medulla. A 50% reduction in the cortical apoptotic nuclear score (ANS) is observed in the EGF-supplemented rats at 48 hours postoperatively when compared with the obstructed and untreated rat cortical kidney (0 #g EGF, ANS = 24.7; 10 #g EGF, ANS = 10.1; 20/a,g EGF, ANS = 9.5; 40/.tg EGF, ANS = 14.8). SimUROLOGY 49 (6), 1997

ilarly, a reduction in the medullary apoptotic nuclear score occurred in the rats supplemented with 20 and 40 #g/day of EGF at 72 hours postoperatively when compared with the obstructed and untreated rat medullary kidney (0 #g EGF, ANS = 43.8; 20 #g EGF, ANS = 13.5; 40 #g EGF, ANS = 29.5). Thus EGF administration to ureterally obstructed rats has a protective effect on the nuclear DNA of renal tubular epithelial cells. EGF SUPPLEMENTATION REDUCES EXPRESSION OF CLUSTERIN

Consistent with the concept of renal cell apoptosis as a genetically active process, certain gene products are highly induced during programmed cell d e a t h ) The SGP-2 or clusterin gene has been frequently associated with apoptosis in such diverse systems as rodent embryonic tissue undergoing programmed cell death, androgen-regulated apoptosis of the rat prostate gland, and hydronephrosis-induced apoptosis of rat renal cells, s'31 Northern blot analysis 2~"for SGP-2 (clusterin) expression was performed on poly(A)+ mRNA isolated from sham-operated control kidneys, obstructed kidneys, and obstructed/EGF supplemented (10 #g/day) kidneys (Fig. 3a). Hybridization of the 32P-labeled cDNA SGP-2 probe revealed the abundant expression of the 2.0 kb transcript for SGP-2 mRNA from the obstructed 977

In situ hybridization for (a) SGP-2 antisense riboprobe on sham-operated control cortical kidney, (b) FIGURE 4. SCP-2 sense riboprobe on obstructed cortical kidney, (c) SGP-2 antisense riboprobe on obstructed cortical kidney, and (d) SGP-2 antisense riboprobe on obstructed cortical kidney treated with 20 pg/day EGF. All kidneys were harvested 72 hours postoperatively. Darker appearing tubular epithelial cells have stained positively for SGP-2 mRNA. Tissues are not counterstained. Photomicrographs are reduced from optical magnification x200.

rat kidney.’ Quantitative densitometry (Fig. 3b) demonstrates a twofold decline in the expression of this transcript at 24 hours and a threefold decline at 72 hours with initiation of daily EGF administration. In situ hybridizationI for SGP-2 expression (Fig. 4) revealed diffuse cytoplasmic staining restricted to the epithelial cells of the distal tubules and collecting ducts in the obstructed kidneys (Fig. 4~). In contrast, sections from the EGF supplemented kidneys (Fig. 4d) show a few regions of diminished hybridization despite persistent dilatation of the distal tubules. These findings confirm that EGF treatment suppresses apoptosis-related gene activity as well as DNA fragmentation. COMMENT Based on past studies of animal models of hyrenal ischemia/reperfusion indronephrosis,x,31 jury,” and aminoglycoside toxicity,33 SGP-2 has proven to be an intriguing biomarker for apoptosis in the kidney. Clearly, this protein is not obligatorily related to apoptosis since it is described as a constitutively expressed gene in tissues such as 978

brain3’ and seminal vesicle and epididymis,‘5-” wherein there appears to be little endogenous apoptosis. The relationship of SGP-2 to apoptosis in the kidney was questioned by the report that early postnatal rat kidneys expressed high amounts of this gene.38 This finding led Harding et a1.38 to postulate that expression of the SGP-2 gene in normal development suggests that SGP-2 plays a role in the differentiation of epithelial structures. However, it is now established that the rat kidney has extensive apoptosis associated with the postnatal maturation of the renal tubules.l’ Given that the adult kidney has little or no expression of this protein, it is still reasonably evident that SGP-2 expression remains tightly associated with periods of apoptosis in this organ. Apoptosis of cells in organ systems other than the kidney is regulated by the local availability of specific growth factors. Probably the best described example of this phenomenon is the demonstrated dependence of motor neurons on neurotrophins synthesized by the peripheral target cell.‘“,t’ During embryonic development, neurons that do not acquire a target cell source of neuro-

trophin undergo cell death. 42 Likewise, hematopoietic cell lineages require the continuous presence of one or more cytokines such as granulocyte-macrophage, colony stimulating factor, or interleukin-2 for their survival. 43-45 Further examples have been described in vitro and include the death of fibroblasts upon basic fibroblast growth factor depletion 12 and the programmed death of cultured embryonic epithelial cells upon EGF withdrawal. 13 Current investigations have revealed that EGF administration prevents apoptotic DNA degradation in cultured kidney embryonic mesenchyme 17 and inhibits apoptosis associated with normal renal development, is These principles have recently taken on a therapeutic importance as investigations have demonstrated the ability of local application of brain-derived neurotrophic factor to rescue motor neurons from axotomy-induced or naturally occurring programmed cell death. 14-t° Such neurotrophic factors that specifically prevent neuronal degeneration and cell death, as well as increase functional activity of targeted neurons, 46 are of significant clinical interest. In the obstructed kidney, the observation that EGF mRNA and protein are reduced at a cellular level before apoptosis of the renal tubular epithelium ~ potentially implicates the decline in EGF expression as the initiator of the apoptotic process in the hydronephrotic kidney. EGF is known to act on renal cells to stimulate their exit from the quiescent Go phase of the cell cycle (noncycling) and enhance their entry into the active cell replication (mitotic) cycle. 47 The mechanism by which EGF supplementation suppresses apoptosis during hydronephrosis is currently unknown. Androgen-regulated apoptosis of prostate cells has been described as a defective cell cycle. ~ Thus, it is possible that EGF supplementation during ureteral obstruction redirects the renal tubular epithelial cell to complete a replicative cell cycle rather than undergoing apoptosis. CONCLUSIONS As the major factors involved in the initiation of apoptotic cell death due to renal obstruction are identified and characterized, the possibility of manipulating the obstructive process becomes feasible. The current investigation demonstrates that subcutaneous EGF supplementation, during the initial period of ureteral obstruction, results in the preservation of nuclear DNA, the suppression of apoptosis-related gene activity, and an increase in renal cortical mitotic activity. These findings are all indicative of enhanced renal tubular epithelial cell preservation and replication. Attention now must be directed toward investigating the renal functional preservation conveyed to the kidney by UROLOGY 49 (0), 1997

EGF supplementation. The present work provides the basis for defining, at a molecular level, the physiologic role of EGF and for exploring its potential utility as an adjunctive treatment during obstructive nephropathy. ACKNOWt.EDGMEN'r.TO Po-Ying Ng and Stephen Savage for technical assistance and to Qais Al-Awqati for comments on the manuscript. REFERENCES 1. Klahr S: Pathophysiology of obstructive nephropathy. Kidney Int 23: 414-426, 1983. 2. Gobe GC, and Axelsen RA: Genesis of renal tubular atrophy in experimental hydronephrosis in the rat. Role of apoptosis. Lab Invest 56: 273-281, 1987. 3. KerrJF, Wyllie AH, and Currie AR: Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. BrJ Cancer 26: 239-257, 1972. 4. Buttyan RE, Zakeri Z, Lockshin R, and Wolgemuth D: Cascade induction of c-fos, c-myc, and heat shock 70K transcripts during regression of the rat ventral prostate gland. Mol Endocrinol 2: 650-657, 1988. 5. Wyllie AH, Morris RG, Smith AL, and Dunlop D: Chromatin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macromolecular synthesis. J Pathol 142: 67-77, 1984. 6. Stanisic T, Sadlowski R, Lee C, and Grayhack JT: Partial inhibition of castration induced ventral prostate regression with actinomycin D and cycloheximide. Invest Urol 16: 19-22, 1978. 7. Martin DP, Schmidt RE, DiStefano PS, Lowry OH, Carter JG, and Johnson EM Jr: Inhibitors of protein synthesis and RNA synthesis prevent neuronal death caused by nerve growth factor deprivation. J Cell Biol 106: 829-844, 1988. 8. Sawczuk IS, Hoke G, Olsson CA, Conner J, and Buttyan R: Gene expression in response to acute unilateral ureteral obstruction. Kidney Int 35: 1315-1319, 1989. 9. Walton G, Buttyan R, Garcia-Montes E, Olsson CA, Hensle TW, and Sawczuk IS: Renal growth factor expression during the early phase of experimental hydronephrosis. J Urol 148: 510-514, 1992. 10. Salido EC, Lakshmanan J, Fisher DA, Shapiro LJ, and Barajas L: Expression of epidermal growth factor in the rat kidney. An immunocytochemical and in situ hybridization study. Histochemistry 96: 65-72~ 1991. 11. Storch S, Saggi S, Megyesi J, Price PM, and Safirstein R: Ureteral obstruction decreases renal prepro-epidermal growth factor and Tamm-Horsfall expression. Kidney lnt 42: 89-94, 1992. 12. Araki S, Simada Y, Kaji K, and Hayashi H: Role of protein kinase C in the inhibition by fibroblast growth factor of apoptosis in serum-depleted endothelial cells. Biochem Biophys Res Commun 172: 1081-1085, 1990. 13. Rawson C, Cosola-Smith C, and Barnes D: Death of serum-free mouse embryo cells caused by epidermal growth factor deprivation is prevented by cycloheximide, 12-O-tetradecanoylphorbol-I 3-acetate, or vanadate. Exp Cell Res 186: 177-181, 1990. 14. Yah Q, Elliott J, and Snider WD: Brain-derived neurotrophic factor rescues spinal motor neurons from axotomyinduced cell death. Nature 360: 753-755, 1992. 15. Oppenheim RW, Qin-Wei Y, Prevette D, and Yan Q: Brain-derived neurotrophic factor rescues developing avian motorneurons from cell death. Nature 360: 755-757, 1992. 16. Sendtner M, Holtmann B, Kolbeck R, Thoenen H, and Barde YA: Brain-derived neurotrophic factor prevents the D79

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