Gene expression in response to acute unilateral ureteral obstruction

Kidney International, Vol. 35 (/989), pp. /315—1319

Gene expression in response to acute unilateral ureteral obstruction IHOR S. SAWCZUK, GERALD HOKE, CARL A. OLSSON, JOHN CONNOR, and RALPH BUTTYAN Department of Urology, Colwnbia University. College of Physicians and Surgeons, 622 West /68th Street, New York City, New York, USA

Gene expression in response to acute unilateral ureteral obstruction.

kidney a rat model of unilateral ureteral obstruction was em-

Acute unilateral ureteral obstruction results in differential growth ployed. This model allowed us to study the molecular signaling characteristics of both the ipsilateral and contralateral kidney. The

associated with compensatory growth in the contralateral kidney, while simultaneously noting the events associated with To evaluate the molecular events occurring in both kidneys after proliferation and death in the ipsilateral kidney. obstruction, we examined the expression of growth related (c-fos. Such an approach might permit a determination of whether c-myc, cH-ras, HSP 70), cell maintenance (/3-actin). and cellular ureteral obstruction results in similar or dissimilar responses of damage (TRPM-2) genes at the mRNA level. In the contralateral kidney an early and transitory induction of cfos and c-,nve expression oc- gene expression in the hydronephrotic versus the contralateral kidney, and whether these changes bear resemblance to those curred while a bimodal induction was noted in the obstructed kidney. The patterns of cH-ras, HSP 70 and actin expression also differed in seen in proliferation, hypertrophy and cell death. With the use both kidneys. Induction of TRPM-2 was noted only in the obstructed of Northern blot techniques we have analyzed and quantitated kidney. Rapid gene activation is evident in both the contralateral and obstructed kidney following unilateral ureteral obstruction. The pat- the expression of mRNA transcripts of several growth related terns of expression are distinct and may reflect the cellular response to (c-jos, c-,nvc, HSP 70, cH-ras), cell maintenance (13-actin), and stress (cell death and stromal proliferation) in the obstructed kidney cellular damage (TRPM-2) genes in both kidneys at various versus a response to a systemic stimulus resulting in cellular hypertrotimes following acute unilateral obstruction. By this method, phy in the contralateral kidney. distinct temporal patterns of gene expression associated with either the obstructed or the hypertrophic kidney were defined. These patterns show that renal cell growth and damage appear Molecular studies of cellular proliferation, hypertrophy and to mimic the growth response of other systems. In addition, we death have demonstrated an orderly pattern of gene expression have identified a gene product (TRPM-2) whose expression associated with these processes. In particular, these studies appears to correlate exclusively with renal atrophy. obstructed kidney undergoes cellular atrophy following an initial phase of interstitial proliferation while the contralateral kidney hypertrophies.

have demonstrated the transient increase in expression of

Methods

certain proto-oncogenes resulting often in a cascade pattern of gene expression [1—5]. The similarity of these patterns suggests that diverse cellular responses may share common signal mech-

Healthy adult Sprague-Dawley rats weighing 200 to 250 g

anisms. Studies utilizing renal models of cell growth and

were housed under standard conditions of heat and humidity on

damage have shown that acute unilateral ureteral obstruction initiates a growth stimulus to both the obstructed and contralateral kidney which results in distinguishable morphologic and biochemical changes. In the obstructed kidney, the predominant growth alterations are due to interstitial hyperplasia. With prolonged obstruction eventual renal atrophy and cellular death occurs [6]. These observations are in contradistinction to those seen in the opposite kidney in which predominately proximal renal tubular cells hypertrophy [7j. Although the phenomenon of renal growth in both the obstructed and contralateral kidney has been extensively studied, the molecular events involved have not been sufficiently pursued. To determine what gene activity might be involved in the

a 12-hour light cycle, with unlimited access to water and a

Received for publication September 6. 1988 and in revised form November 28, 1988 Accepted for publication January 12. 1989

natant was removed and the RNA-rich pellet was dissolved and phenol/chloroform extracted. Polyadenylated [poly (A) J mRNA was isolated by oligodeoxythymidine affinity chromatography and quantitated by spectrophotometry at 260 nm [9].

© 1989 by the International Society of Nephrology

Unilateral ureteral obstruction model

standard diet. Using sodium pentobarbital anesthesia (0.04 mg/ kg intraperitoneally) animals underwent left proximal ureteral ligation using a silk suture under sterile conditions. The animals were sacrificed at 15 minute intervals until 1 hour, at 10, 24, 48

and 168 hours postoperatively. Normal and sham operated kidneys were analyzed for control purposes. Kidneys were rapidly removed, frozen in liquid nitrogen and stored at —70°C until RNA extraction was performed.

,nRNA extraction and Northern blotting hybridization The frozen surgical specimens were pulverized and homoge-

nized in 5% 2-mercaptoethanol/guanidine thiocyanate solution. Samples were then ultracentrifuged in cesium chloride (1.72 mgI ml density) at 25.000 RPM (20°C) for 24 hours [8]. The super-

1315

1316

Sawczuk et a!: Gene activation ftuloti'ing uretera! obstruction A

N

10 24 48 168 hours

1

c-fos

2.2 kb

c-myc

2.4 kb

Contralateral

Obstructed

A N

30 45 60 N

15

15

30

60

45

C-

c-fos

2.2 kb

1.2kb

cHa-ras

c-myc

2.4 kb

HSP-70

2.4 kb

2.0 kb

Ribosomal B

Contrslateral kidney

Obstructed kidney 6000

1.2kb

Actin

5

2000

4500 1500

3000 1000

2.0kb

Ribosomal

c-fos

10000

7500

4O00

15000

-

—

e -*

4

60

45

Time, minutes

30

45

60

Time. minutes

cHa-ras

60000

ferred to nitrocellulose filters in 20 x SSC (I x SSC = 0.15 M

45000

sodium chloride/0.0l5 M sodium citrate) for Northern blot analysis [10].

•0 '5

0

500

30

Fig. 2. Gene expression in both kidneys under I hour. Northern blot and densitometric analysis of c-,nyc and cfos gene expression in both the obstructed and contralateral kidney following complete unilateral ureteral obstruction from 0 to 60 minutes.

8000

U, C U,

0

1000

500

c-myc

12000

30000

1500

1000

/T\

—

16000

Ot

1500

15

20000

U,

0 0 0

—

0C 2U

5

4

5000 2500

75000

500

U

B 12500

U,

5 1500

c-fos

HSP7O

37500 30000

Purified DNA fragments were labeled with 32P by nick translation [II]. Typical incorporation procedures yielded probes with specific activities of 3 to 8 x 106 DPM/g DNA. Denatured 32P, nick-translated eDNA gene probes were then

22500 15000

hybridized with the mRNA on nitrocellulose filters at 42°C overnight. Filters were washed to a final stringency of 0.1 x

7500

SSC at 60°C. Autoradiograms were prepared by exposing the actin

12500 10000

hybridized filters to X-omat'm AR film. To employ the same mRNA filters for studying the expression of several genes, hybridized probes were removed from the filters for subsequent re-hybridization by incubation at 100°C in diethylpyrocarbonate treated water. Prior to subsequent hybridization, autoradiogra-

7500 5000 2500

Ni

10

24

48

168

Time, hours

Fig. 1. Serial changes in gene expression in the contraluieral kidney following unilateral ureteral obstruction. Poly (A) mRNA 10 .tg was extracted at varying times from I hr to 168 hrs from the contralateral kidney following complete unilateral ureteral obstruction. The RNA was electrophoresed, blotted to nitrocellulose filters, hybridized to a series of 32p-labeled probes to detect specific gene transcript levels and

autoradiograms were obtained. Band densities were quantitated on a Joyce-Loebel Chromosome 3 densitometer and corrected against l8S ribosomal RNA.

phy was again performed to insure adequate removal of the previously hybridized probes. Finally, quantitative densitometry was per-formed on autoradiograms using a computer based

measurement of the integral of area and density on a JoyceLoebel Chromoscan 3 densitometer. An internal ribosomal standard was determined by densitometric analysis of ribo-

somal bands in order to detect variations in RNA loads. Differences in mRNA loading were corrected against the l8S ribosomal RNA standard to correct for these variations.

cDNA probes The DNA probe for c-los was obtained from Thomas Curran

Poly (A mRNA (10 .tg/sample) was electrophoresed on denaturing 0.8% agarose/2.2 M formaldehyde gels and trans-

(Roche Institute of Molecular Biology, Nutley, New Jersey, USA) murine c-inv (third exon) from Frederick Alt (Columbia

1317

Sawczuk et a!: Gene activation following ureteral obstruction

University, New York, New York, USA); cH-ras from the BS9 plasmid; mouse HSP 70 related cDNA (PMHS2I3) from Larry Moran (University of Toronto, Toronto, Ontario, Canada); beta actin from Donald Cleveland (Johns Hopkins University, Baltimore, Maryland, USA) and TRPM-2 from Martin Tenniswood (University of Ottawa, Ottawa, Ontario, Canada). Control for the integrity and amount of mRNA was determined by an 18S ribosomal RNA probe, obtained from Ramreddy Guntaka (University of Missouri, Columbia, Missouri, USA).

N 11024 48WkS

A c-fos

c-myc

2.4kb

cHa.ras

1.2kb

S

Results

These experiments were divided into early and late time intervals (0 to 60 minutes and 1 hour to 168 hours). The major c-fos mRNA was detected as a 2.2 kb species; c-myc was detected as a 2.4 kb species, cH-ras as a 1.2 kb species, HSP 70 as a 2.4 kb species, /3-actin as a 1.2 kb species, and TRPM-2 as a 2 kb species. Gene expression in the con tralateral kidney

many of the classical studies of compensatory renal hypertrophy had demonstrated biochemical alterations associated with the growth process as early as 24 hours following contralateral renal damage or deprivation, we had presumed that initiating our studies of gene expression by the one hour interval would be sufficient to detect the molecular events associated with compensatory growth (Fig. I). By one hour Since

there was a threefold increase in cH-ras and HSP 70 expression with a peak at 10 hours. A steady state of a twofold increase in expression of these genes was then maintained from 24 hours to one week. Actin expression also paralleled that of cH-ras and

HSP 70. Increased expression of c-fos and c-myc was not detected. This apparent absence of expression of the genes encoding DNA binding proteins was in sharp contrast with what had been observed in similar studies stimulating the growth of

cells in vitro. However, the observed induction of c-Jos and c-myc genes in proliferative cellular growth in culture was usually quite immediate and transient. Therefore, earlier time intervals following unilateral ureteral obstruction were studied. Figure 2 shows, in serial fashion, the relative expression of the same genes in the contralateral kidney within 15 minutes to one hour following unilateral obstruction. What can be immediately

appreciated is that those genes encoding for DNA binding proteins (c-fos and c-myc) were very prominently expressed during the initial hour following contralateral ureteral ligation. Within 15 minutes after ureteral occlusion, there was an induction of both c-fos and c-myc, which however was transient, returning to baseline levels by one hour. Induction of c-fos and c-,nyc transcripts were not detected in sham operated animals during any of the time intervals. However, variations in expression of cH-ras, HSP 70 and actin were

detected in the sham operated animals during the one hour period. Whether these variations are due to the stress of the surgical procedure or to the anesthesia is not known and it is difficult to speculate on their role during this phase. No alterations in gene expression were detected in the sham operated

2.2 kb

-

ed

HSP-70

2.4kb

Actin

1.2kb

Ribosomal

2.0 kb

B 12000 9000 6000 3000

20000 15000

10000 5000 'I, C

30000

2 22500 U

15000 7500

20000 15000 10000

5000

20000 15000 10000

5000

Ni

10

24

48

168

Time, hours

Fig. 3. Serial c/lan ges in gene expression in the obstructed kidney. Ten micrograms of poly (A) mRNA was extracted from the obstructed

kidney following acute ureteral obstruction from I to 168 hrs. The mRNA was electrophoresed. transferred to nitrocellulose filters and hybridized to 3p-labeled probes. Autoradiography and band densitometry was performed.

animals after one hour. Gene expression in the obstructed kidney

Induction of c-fos and c-tnyc transcripts occurred IS minutes following ureteral obstruction (Fig. 2). In the case of c-rnyc, this

expression was quite transient, similar to that seen in the contralateral kidney, whereas in the case of c-fos, there was continued expression at the one hour interval, suggesting a prolonged function of c-fos in the obstructed versus the contra-

1318

Saw czuk et al: Gene acln'aru,n following ,retera1 obstruction

A I

/

Obstructed kidney

r

Contralateral kidney

B

Contralateral kidney

,'

8000

2kb

5 0

4000

C C

TRPM-2

TRPM-2

Obstructed kidney 16000 12000

8000

Fig. 4. Analysis of TRPM-2 expression in the obstructed and contralareral kidneys. TRPM-2 gene expression in both the obstructed and contralateral kidney was examined

from 1 to 168 hrs. The blots used in Figs. 1 & 2 were stripped of probe and then rehybridized with the TRPM-2 probe. An induction of TRPM-2 was noted only in the obstructed kidney.

4000 10

24

48

168

Time, hours

lateral kidney. After the initial hour there was a continued and sequential expression of proto-oncogenes and heat shock gradual decline of c-fos expression up to 24 hours and a genes [3, 12]. The molecular events that accompany cetlular continued low level of c-myc' expression over the same time interval, following which there was an apparent major reinduction of both genes by 48 hours (Fig. 3). Actin also exhibits an increased expression at 24 hours following ureteral obstruction while HSP 70 maintains a steady state. Declines in expression

are seen at 48 hours in the case of HSP 70 and actin while cH-ras appears to undergo a secondary re-expression at the one week period.

hypertrophy remain relatively undefined. It is of interest, however, that an induction of both c-fos and c-myc has already been found to be an early event associated with cardiac hypertrophy produced by pressure overload [4] as well as in CRG following unilateral nephrectomy [13]. An early induction of c-fos/c-rnyc activity in these systems is strikingly similar to that seen during

proliferative stimulation of cultured cells and during liver regeneration. The molecular events that accompany cellular

Of even greater interest are the sequential alterations of damage or death are more poorly defined. That cellular activity occurs with cell death has been noted as a general increase in RNA synthesis or as the induced expression of specific gene obstruction and by 48 hours the TRPM-2 transcript could be products during the death phase of cells [14]. The importance of estimated to make up 0.5% of the total mRNA of the kidney, a this activity during cellular death has been strengthened by minimum 17,000-fold induction, as determined from densito- experiments in which either RNA or protein synthesis inhibimetric analysis of the Northern blot autoradiogram. TRPM-2 tors can delay cell death or damage [IS]. Recent analysis of a hormonally-dependent cell death system, regression of the rat mRNA was not detected in the normal kidney, sham operated animals, contralateral kidney nor induced after androgen depleventral prostate gland following castration, has indicated that tion in the kidney at any of the time points. transcripts encoding c-fos, c-,nyc and HSP 70 are sequentially TRPM-2 gene expression in the obstructed kidney (Fig. 4). This message was initiated as early as 10 hours following ureteral

Discussion

induced similar to that during cellular proliferation [5]. The induction of such a reactive cascade during different cellular

Cells respond to environmental stimuli through alterations in their pattern of gene expression. This has been studied most extensively in vitro for the process of cell proliferation. Shortly

processes suggests that diverse cellular responses may share common signal mechanisms.

after the addition of proliferative-stimulating substances to

characterization of distinct molecular changes associated with hydronephrotic renal damage or with hypertrophic renal growth in a single in vivo model system. The similar initial induction of c-fos and c-rn ye in both the obstructed and contralateral kidney appears to be an early event in the response to acute obstruc-

quiescent cells in culture, a series of gene products are induced in a defined temporal order. The earliest event is often an abrupt and transient induction of mRNA and protein products encoded by the c-fos gene [1]. This is followed by a transient increase in gene products encoded by c-rnvc and even later, by increased activity of the actin, heat shock 70K gene and ms related genes

[2]. The ability to stimulate tissue proliferation in vivo (that is, during liver regeneration) is associated with a similar transient

The use of a rat ureteral obstruction model permitted a

tion and may possibly play a key role in the initiation of the proliferative, hypertrophic and damage responses in this model. The molecular changes seen in the contralateral kidney follow-

ing unilateral ureteral obstruction mimic those seen in cell

1319

Sai'czuk et a!: Gene activation following ureteral obstruction

Reprint requests to ihor S. Sawczuk, Department of Urology, Goculture systems. There is a rapid and transitory induction of University, College of Physicians and Surgeons. 622 West 168th c-fos and c-myc beginning within 15 minutes of contralateral lu,nh,a Street, New York City, New York /0032, USA.

obstruction and terminating by one hour. Commencing with the induction of these genes there are also alterations of the other genes probed, which continue to demonstrate increased expres-

sion beyond the first hour, extending for the duration of the experiment (1 week). In contrast, the obstructed kidney shows two major initiating signals. The initial induction of these genes in the obstructed kidney may again represent a common activation event during

the hyperplastic response. Of greater interest is the second induction phase of c-fos and c-myc coincident with TRPM-2 induction. TRPM-2 (testosterone-repressed prostate message)

was initially thought to represent an example of an androgen dependent protein expressed in the prostate alone after castration [16]. Subsequently it was found that this gene is induced during cellular damage in other non-androgen dependent systems [17]. The protein product and the function of the TRPM-2 gene is unknown; however, because of its relationship to tissue

regression certain hypotheses may be considered. For one, TRPM-2 may be activated in an attempt to protect cells against

the noxious conditions which induce their damage. Alternatively, it may be involved in either the mechanism by which cells are damaged or serve as an attractant factor to lymphocytic cells in an attempt to rid the tissue of damaged cells. Further

experiments to understand the role of TRPM-2 during renal damage are currently underway.

We interpret our results as demonstrating the differential pattern of sequential gene expression associated with both renal

growth and renal damage. Rapid gene activation is evident in both the contralateral and obstructed kidney following unilateral ureteral obstruction. The patterns of expression are distinct and may reflect the cellular response to stress (cell death and stromal hyperplasia) in the obstructed kidney versus a response to a systemic stimulus resulting in cellular hypertrophy in the

contralateral kidney. It is of interest that both a noxious stimulus (hydronephrosis) and presumed growth stimulus (re-

sulting in hypertrophy) initially activate identical gene cascades. The above hypothesis must be further studied with experiments oriented to the application of in situ hybridization techniques which would permit the molecular events in each subpopulation of cells within the kidney to be characterized.

Acknowledgments

ISS is an American Urological Association Scholar and is supported by a National Kidney Foundation Young Investigator Grant.

References

I.

MULLER R, BRAVO R, BURCKHARDT J, CURRAN 1: Induction of c-fos gene and protein by growth factors preceeds the activation of c-mvc. Nature 314:546, 1984 2. CURRAN T, BRAVO R. MULLER R: Transient induction of c-fos and c-mvc is an immediate consequence of growth factor stimulation. Cancer Survey 4:656, 1985 3. THOMPSON NL. MEAD JE, BRAUN L, GOYETTER M, SHANK PR, FAUSTO N: Sequential protooncogene expression during rat liver regeneration. Cancer Res 46:3 III, 1986

4. 1zwio S. NADAL-GINARD B. VIJAK M: Protooncogene induction and reprogramming of cardiac gene expression produced by pressure overload. Proc Nat! Acad Sci USA 85:339, 1988 5. BUTTYAN R, ZAKERI Z, L0cK5HIN R, WOLGEMUTH D: Cascade induction of c-los, c-mvc and heat shock 70K transcripts during regression of the rat ventral prostate gland. Mo! Endocrino! 2:650, 1988

6. NAGLE RB. BULGER RE, CUTLER RE. JERvI5 HR. BENDITT EP:

Unilateral obstructive nephropathy in the rabbit. I. Early morphologic, physiologic and histochemical changes. Lab invest 28:4265, 1973

7. PAULSON DF, FRALEY EE: Compensatory renal growth after unilateral ureteral obstruction. Kidney mt 4:22, 1973 8. CHIRGWIN JM. PRZYBYLA AE, MACDONALD PJ, RUTTER Wi: Isolation of active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294, 1979

9. Aviv H, LEDER P: Purification of biologically active globulin messenger RNA by chromatography on oligo thymidyllic acidcellulose. Proc NatI Acad Sci USA 69:1408, 1972 10. LEHRACH H. DIAMOND D, WOZENY JM, BOEDTKER M: RNA molecular weight determination by gel electrophoresis under denaturing conditions. A critical re-examination. Biochemistry 16:4743, 1977

II. RIGBY PWJ. DIECKMAN R, RHODES C, BERG P: Labeling of deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase. mt i Mo! Biol 13:237, 1978 l2. CARR BI. HUANG TH. BUzIN CH, ITAKURA K: Induction of heat shock gene expression without heat shock by hepatocarcinogens and during hepatic regeneration in the rat liver. Cancer Res 46: 5106. 1986 13. REITELMAN C. OLSSON CA, BUTTYAN R, ALT FW. NGUYEN Huu MC. SAWCZUK IS: Gene expression during post natal and compen-

satory renal growth. (abstract) J Uro! 137:2tOA, 1987

14. LEE C: Physiology of castration-induced regression in the rat prostate. Progr Cliii Biol Res 75A: 145. 1981 IS. CONNOR J, SAwCZUK IS. BENSON MC, TOMASHEFSKY P. O'Tooi KM. OLssoN CA, BUTTYAN R: Calcium channel antagonists delay regression of androgen-dependent tissues and suppress gene activ-

ity associated with cell death. Prostate 13:119, 1988 16. MONTPETIT ML. LAWLESS KR. TENNISWOOD M: Androgen repressed messages in the rat ventral prostate. Prostate 8:25, 1986 17. BUTTYAN R. OLssoN CA. HOKE G, NG PY. TOMASHEFSKY P. SAWCZUK IS: TRPM-2. a unique gene whose expression correlates

with degenerative atrophy of normal and cancerous tissue. (abstract) J Uro! l39:l92A. 1988