Elevated Proto-Oncogene Expression in Polycystic Kidneys of the C57BL/6J (cpk) Mouse”2’3

Benjamin D. Cowley, Jr., M.D., Laurie J. Chadwick, M.S., Jared J. Grantham, M.D., and James P. Cabvet, Ph.D.4

oncogene expression later in disease progression or B.D. Cowley, J.J. Grantham, Department of Medicine that there is a secondary response in the kidney to (Nephrology). University of Kansas Medical Center, the progressive renal failure. Kansas City. KS Key Words: c-fos. c-, c-ki-ras, renal , kidney failure B.D. Cowley, L.J. Chadwick, J.P. Calvet, Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS T he polycystic kidney diseases (PKD) are a group (J. Am. Soc. Nephrol. 1991; 1:1048-1053) of disorders characterized by massive kidney enlargement due to the development of epithellal- lined cysts derived from nephrons and collecting

ducts ( 1 ). PKD can be inherited (2) or provoked by ABSTRACT environmental factors (3,4). Several pathological Polycystic kidney disease in the C57BL/6J (cpk) findings suggest an abnormality of cellular growth mouse is an autosomab recessive disorder which control in PKD. First, as cysts grow, increased num- beads to the rapid development of renal cysts and bers of cells are produced to line cavities (5). kidney failure during the first 3 to 4 postnatal weeks. Second, undifferentiated cells, cell overgrowth, and Previously, we showed that the cystic kidneys of af- pobypoid structures have been noted in the epithe- fected mice have abnormally elevated levels of c- hum lining renal cysts (5,6). Third, an increased myc mRNA. In the study presented here, it is shown incidence of renal tumors has been documented in that mRNAs for the proto-oncogenes c-fos and c-Ki- some forms of renal cystic disease (7-9). The regulation of cell proliferation is currently ros, as well as c-myc, are markedly elevated in cystic thought to involve alterations in the expression of kidneys, suggesting that there is a more general several proto-oncogenes. Specifically, elevated proto- abnormality in expression associated with the oncogene expression has been demonstrated in em- disease. It is also evident that there are two stages bryonic and neonatal tissues ( 1 0), in cultured cells to this abnormal proto-oncogene expression. In the stimulated with (1 1 - 1 4), in regenerating first stage, which occurs up through the second post- liven after partial hepatectomy on toxic injury (15,16), natal week, there are modest increases in proto- in regenerating kidney after toxic injury (1 7), and in oncogene mRNA which parallel the increased cell a number of malignant tumors (1 8, 1 9). In particular, proliferation that accompanies cyst growth at this the proto-oncogenes c-fos, c-myc, c-Ki-ras, and c-Ha- time. In the second stage, which occurs after the ras have been associated with the transition of cells second postnatal week, there are markedly ebe- from G0, through G1 , to S phase. We previously demonstrated markedly elevated vated bevels of proto-oncogene mRNA that are seen mRNA levels for the proto-oncogene c-myc in the at a time when cell proliferation is declining. The kidneys of a mouse model of autosomal recessive PKD development of this latter stage suggests either that (20). In these mice, there Is marked kidney enlarge- there is a fundamental abnormality intrinsic to poly- ment, due primarily to the development of fluid-filled cystic kidneys that leads to uncontrolled proto- cysts, but also due to a modest increase in renal cell mass. Because the overall rate of cell proliferation in these cystic kidneys, as measured by several param- I Received May 9, 1990. Accepted October 23. 1990. ), 2 A preliminary account of these results appeared in abstract form (Cowley, eters (2 1 was not increased to the same degree as BD, Jr. Grantham JJ, Calvet JP: Elevated proto-oncogene expression in poly- the increase in c-myc mRNA, we suggested that the cystic kidney disease (Abstract). FASEB J 1988;2:A839). expression of this proto-oncogene was abnormally 3 The manuscript was sent to Dr. Frank Carone. Guest Editor, for review by expert high relative to the increased rate of cell division. referees, for editorial decision, and for final disposition.

4 Correspondence to Dr. J.P. Calvet, Department of Biochemistry and Molecular These results led us to evaluate the expression of Biology, University of Kansas Medical Center, Kansas City, KS 66103. several other proto-oncogenes associated with cell 1046-6673/0 108-1 048$03.00/0 proliferation. We now report that the bevels of c-fos Journal of the American society of Nephrology Copyright C 1991 by the American Society of Nephrology and c-Ki-ras mRNAs, as well as that of c-myc mRNA,

1048 Volume I ‘ Number 8 ‘ 1991 Cowley et 01

are abnormally elevated in the cystic kidneys of lOx Denhardt’s sobution-25O g of tRNA pen mL-20 C57BL/6J (cpk/cpk) mice, particularly at end-stage, mM sodium phosphate buffer (pH 7.8)- 1 0% dextran indicating ( 1 ) that there is a more general abnormal- sulfate, with 1 06 cpm of probe per mL of hybnidiza- ity in the expression of associated with cell tion solution. Filters were washed at 66#{176}CIn 1 x SSC- proliferation and (2) that there are two stages to the 0.1% SDS for 1 h, O.3x SSC-O.1% SDS for 1 h, and abnormal proto-oncogene expression over the course 0. 1 SSC-0. 1 % SDS for 1 h (20x SSC is 3 M NaCl-O.3 of cyst growth and disease progression. M trisodium citrate; pH 7). Filters were then exposed to Kodak XAR film. Autoradiographs were quantl- tated by densitometry; values presented in Table 1 METHODS represent the results for all Northern hybridizations. Transcriptions for generation of 32P-nadiobabeled Animals anti-sense RNA probes specific for c-fos, c-myc, c- C57BL/6J (cpk) mice (22) were maintained as a Ki-ras, and c-Ha-ras were performed as previously breeding colony in our facilities. Homozygous neces- described (1 7). Briefly, the used for gener- sive cpk/cpk mice show a uniform progression of ation of probes were: ( 1 ) pfos, containing a 1 .8-kbp renal cystic changes that include both proximal tu- EcoRI/SstI fragment from the mouse c-fos gene sub- bule and collecting duct dilations at 1 week of age, cloned into pSP64 (Promega, Madison, WI). This plas- followed by rapid development of collecting duct cysts mid was linearized with EcoRI before transcription; at 2 and 3 weeks of age (2 1). These mice develop (2) pc-myc, containing a HtndIII/SacI fragment from massively enlarged cystic kidneys, become azotemic, exons 2 and 3 of mouse c-myc subcboned Into pSP64. and die from renal failure usually soon after 3 weeks This plasmld was linearized with PvuII before tran- of age. Infrequently, an affected mouse will live as scniption; (3) pBvHR, containing an 800-bp SacI/Pstb long as 5 or 6 weeks. Cystic mice were sacrificed at fragment of the v-Ha-ras gene subcboned Into pGEM-

1 , 2, 3, and 5 weeks of age, and kidneys were proc- 3Z (Promega). This plasmld was linearized with essed for RNA isolation. Numerous animals, usually EcoR! before transcription; and (4) pBKI-Ras, con- from several litters, were evaluated at each time taming a 52O-bp EcoRI/PstI fragment from a human point, except for a single 5-week-old animal. Noncys- c-KI-ras cDNA subcboned into pGEM-4Z. This plas- tic littenmates served as controls. mid was linearized with PstI before transcription. All transcriptions were carried out In the presence of a- 132P]guanoslne 5 ‘-tniphosphate (DuPont/NEN Re- RNA Isolation and Hybridization search Products, Boston, MA) with SP6 RNA polym- erase (Promega), and specific activities for all probes All cystic kidneys and all noncystic kidneys from were calculated to be approximately 1 O cpm/g. Re- each bitten analyzed were separately pooled. Whole sults presented are representative examples of, In kidney RNA was isolated by the method of Chirgwin most cases, multiple experiments. The following is a et at. (23), and the samples were enriched for poly(A) summary of the numbers of experiments carried out. RNA by one cycle of oligo(dT)-cellubose chromatogna- c-fos: 1 week, 1 litter/i blot; 2 weeks, 5 littens/9 phy. RNA samples were denatured in 2.2 M formal- blots; 3 weeks, 8 litters/lO blots; 5 weeks, 1 animab/ dehyde-50% formamide and electnophoresedin 2.2 1 blot; c-myc: 1 week, 1 litter/i blot; 2 weeks, 5 M formaldehyde- 1 .5% aganose gels. All gels were bittens/9 blots; 3 weeks, 8 litters/i5 blots; 5 weeks, 1 stained with acnldine orange to locate the 1 8S and animal/i blot; c-Ki-ras: 1 week, 1 litter/i blot; 2 285 nlbosomal RNA bands to verify the integrity of weeks, 2 bitters/2 blots; 3 weeks, i lItter/2 blots. the RNA samples and to confirm that equal amounts of RNA were loaded in each gel lane. The stained gels were photographed and the 1 85 and 285 nRNAs were used as molecular weight markers to confirm that TABLE I . Relative increase in proto-oncogene the probes hybridized to mRNAs of appropriate size. mRNAs in cystic compared with normal kidneys of RNA was then transferred to nitnocelbulose and hy- C57BL/6J (cpk) mice bnidized as described previously (1 7,20). Filters were baked overnight at 60 to 70#{176}C. Inc rease at age Filters were prehybnidized at 66#{176}Cin 3x SET (20X (weeks): SET is 3 M NaCl-0.04 M EDTA-0.6 M Tnis-HC1; pH 8)-0.1% SDS-lOx Denhardt’s solution (lOx Den- 2 3 handt’s solution is 0.2% Ficolb-0.2% Pobyvinylpynoli- done-0.2% bovine serum albumin) for 1 h and then c-fos -3x >lOx in 3x SET-0. 1 % SDS- 1 Ox Denhandt’s sobution-250 c-myc 2-6x 25-30x Lg of tRNA per mL for 1 h. Hybridizations were c-Ki-ras -4x >lOx performed overnight at 66#{176}Cin 3x SET-O. 1 % SDS-

Journal of the American Society of Nephrology I 049 Proto-oncogene Expression in PKD

RESULTS A 1 2 3 5 We previously demonstrated markedly elevated c- wk wk wk wk myc mRNA levels In the kidneys of 2- and 3-week- old C57BL/6J (cpk/cpk) cystic mice (20). Although NL CV NL CY NL CV CV these polycystlc kidneys become abnormally en- barged, much of this increase in size is due to the f o s -0’. development of large fluid-filled cysts. Thus, whereas the overall rate of cell proliferation in these cystic

kidneys Is increased compared to normal (20,2 i ), this increase is much less than the increase in c-myc m y C -0” mRNA levels. As such, c-myc expression is dispro- portionateby high relative to the rate of cell division. These results prompted us to evaluate the expression of other proto-oncogenes during the development of cystic disease. 1 8 S -0” Figure i shows c-fos and c-myc mRNA levels in total and poly(A)’ RNA from kidneys of normal and

cystic mice at 1 to 3 weeks of age. We were also able B to evaluate these mRNAs in a single cystic mouse 2 3 2 3 that survived to 5 weeks of age. In total RNA (Figure w k vk__ wk w k 1 A) from normal kidneys, c-fos levels were barely NL CV NL CV NL CV NL CV detectable. However, c-fos was easily detectable in total RNA of cystic kidneys at i week of age, was somewhat increased at 2 weeks, and was highest at 3 weeks of age. c-myc mRNA levels were apparent even In normal kidneys at 1 . 2. and 3 weeks of age. fos-0” myc-o’ There was a clear increase in c-myc mRNA in cystic as compared with normal kidneys at 1 week. As we reported previously (20), there was also a significant increase at 2 weeks and a further, very marked in- crease at 3 weeks of age. These elevations in c-fos and c-myc mRNAs were maintained in cystic kidneys through 5 weeks of age. Figure 1 . Levels of c-los and c-myc mRNAs in polycystic Figure lB shows c-fos and c-myc mRNA levels in kidneys. (A) RNA blot hybridization with 5 zg of total kidney poly(A) RNA. Faint bands of c-fos mRNA were vlsi- RNA per lane from normal (NI) and cystic (CV) C57B1/6J ble in the poby(A) RNA of normal kidneys at 2 and 3 (cpk) mice at I , 2, 3, and 5 weeks of age. (Top) c-los mRNA, (middle) c-myc mRNA, and (bottom) 18S rRNA stained with weeks, and significant increases were apparent in acridine orange and photographed before transfer to con- the cystic kidneys at these same ages. c-myc mRNA firm that equal amounts of RNA were loaded in each gel was also increased in the poly(A) samples from cys- lane. The relative migrations of 18S and 28S rRNA5 were tic kidneys at 2 and 3 weeks of age. Densitometry of used to confirm the identification of the proto-oncogene the autoradiognams (Table 1) showed that the in- mRNAs. (B) RNA blot hybridization with 5 g of poly(A) RNA creases In c-fos mRNA were approximately threefold per lane from the kidneys of NI and CV mice at 2 and 3 at 2 weeks and greater than i 0-fold at 3 weeks of weeks of age. (Some cross-hybridization of the probes to age. The increases in c-myc mRNA were approxi- I 85 rRNA can be seen below the c-los and c-myc bands in mateby fivefold at 2 weeks and 25- to 30-fold at 3 total RNA. This is absent in the poly(A) RNA.) weeks of age (20). c-fos and c-myc encode DNA-binding , was also found to be elevated at 3 weeks, although thought to be important in the regulation of gene this increase was only about twofold (data not expression (24-29). To investigate another class of shown). For all proto-oncogenes examined, the bang- pnoto-oncogenes, c-Ki-ras and c-Ha-ras mRNA levels est differences between normal and cystic kidneys were determined. Figure 2 shows the levels of c-Ki- were during the most severe stage of the cystic dis- ras mRNA in total RNA from normal and cystic kid- easejust before complete renal failure. neys at 2 and 3 weeks of age. As with c-fos and c- myc, c-Ki-ras showed a significant elevation at 2 DISCUSSION weeks and a more marked increase at 3 weeks. These increases were approximately fourfold at 2 weeks The PKDs are a clinically important and frequently and 10-fold at 3 weeks (Table 1). c-Ha-ras mRNA encountered form of renal disease. The most common

1050 Volume I ‘ Number 8 ‘ 1991 Cowley et al

2w 3w constitutive bevels in transformed cells in culture and in malignant cells (1 8, 1 9). In either case, cell prolif- NLCY NLCY eration and oncogene expression appear to be cou- pled. This would also seem to be the case in the early 1234 stage of cystic disease In the cpk mouse but not in the batter stage. At 1 and 2 weeks of age, there is a slight increase in the overall rate of cell proliferation

in cystic kidneys (2 1 ). Coincident with this, there are slight increases in the bevels of the -regu- bated mRNAs, histone H4 (20,2 1) and fl-actln (33) and modest Increases in the bevels of the proto-oncogenes evaluated in this study (Table 1). These results are consistent with the Idea that proto-oncogene expres- Ki-ras. sion at 1 and 2 weeks Is largely a manifestation of cell proliferation. In comparing normal and cystic kidneys at 3 weeks of age, there continues to be only a minimal increase in the overall rate of cell probif- enation and in the expression of the histone H4 and /3-actin genes (20,2 1 ,33); however, there Is markedly elevated expression of the c-fos, c-myc, and c-KI-ras proto-oncogenes. This Is demonstrated In Figure 3,

3.

Figure 2. levels of c-Ki-ros mRNA in poly(A) kidney RNA. 0) RNA blot hybridization with 5 g of poly(A) RNA per bane > from the kidneys of normal (NI) and cystic (CV) mice at 2 . 2 and 3 weeks of age. This autoradiograph was intentionally overexposed to demonstrate the low but detectable bevel z of c-Ki-ras mRNA in normal kidneys.

type, autosomal dominant PKD, accounts for appnox- imately 7 to 10% of the dialysis population in the United States (30). Acquired PKD is frequently seen in patients with end-stage renal disease and is a growing concern because of the now-recognized as-

. sociation with renal malignancies (7,3 1 ). Several hy- !; potheses have been suggested for the pathogenesis of the renal lesions seen in PKD, including abnor- malities of tubular basement membrane, tubular ob- < -. .- -. \ struction, and abnormalities of tubule cell prolifera- z . ‘S. .% tion (32). Regardless of the primary etiology, It is clean that increased numbers of cells are required during E cyst formation (5), suggesting that alterations in the expression of genes associated with cell proliferation may accompany cyst growth. We previously demon- 0 - - strated abnormally elevated bevels of c-myc mRNA in 1 2 3 the kidneys of pobycystic mice (20). In the current Age (weeks) study, we demonstrate that this abnormal proto-on- cogene expression is not limited to c-myc but also Figure 3. Disproportionate expression of the c-myc and his- tone H4 genes. c-myc (upper panel) and histone H4 (bower involves the c-fos and c-Ki-ras genes. panel) mRNA levels in normal (closed symbols) and cystic All of these proto-oncogenes have been shown to (open symbols) kidneys of C57B1/6J (cpk) mice based on be expressed in a transient, cell cycle-specific man- densitometry of autoradiograms. Ordinates represent arbi- ner In normal proliferating cells in vitro (1 1-14) and trary densitometric units. c-los and c-Ki-ras showed similar in vivo ( 1 5- 1 7). In contrast, these same genes (or patterns to that shown for c-myc. Values for H4 are from an their netrovinab homobogs) may be expressed at high, autoradiogram published in reference (21).

Journal of the American Society of Nephrology I 051 Proto-oncogene Expression in PKD

which graphically illustrates that although the level marker for cell proliferation. However, we can not of histone H4 mRNA falls between 2 and 3 weeks of determine at this time what robe the abnormally high age in both normal and cystic kidneys, the level of c- proto-oncogene bevels may play in the pathogenesis myc mRNA actually increases (c-fos and c-Ki-ras of PKD. In this context, it will be important to deter- show similar patterns), suggesting that at 3 weeks of mine if it is the cysts alone, or if it is other nephron age there is a disassociation of cell proliferation and segments as well, that are responsible for the ele- proto-oncogene expression. Although affected cpk vated proto-oncogene mRNA levels. In this regard, it mice experience a progressive boss of kidney function is of interest to note the development of PKD in during the course of their disease, serum urea nitno- transgenic mice expressing the simian virus 40 lange gen does not reach critically high bevels until 3 weeks T antigen (37) and the c-myc oncogene (38). These of age (20,21). Despite this, it does not appear that observations, viewed in conjunction with the data the abnormally high proto-oncogene expression is a presented here, suggest that abnormal growth regu- systemic response to the azotemia, because increased lation in general and pnoto-oncogene expression in bevels of proto-oncogene expression were not found particular may be involved in the pathogenesis of in liver and salivary gland (data not shown). The PKD. distinctly different patterns of proto-oncogene expression early and late in the disease suggest that ACKNOWLEDGMENTS at least two processes are responsible for the eleva- tion of proto-oncogene mRNA levels. However, We thank B. Magenheimer and V. Donoso for excellent technical whether the increases in proto-oncogene expression assistance and G. Andrews for construction of the c-myc and c-fos plasmids. Computer resources used to carry out our studies were in cystic kidneys reflect an intrinsic abnormality provided by the BIONET National Computer Resource for Molecular related to the pathogenesis of the disease on represent Biology. which was funded by the Biomedical Research Technology a kidney-specific response to the renal failure is not Program. Division of Research Resources. National Institutes of yet clear. Health, grant number P41RR01685. This work was supported by NIH grants DK37100 to J.P.C. and DK13476 to J.J.G. and a grant The disassociation of cell proliferation and proto- from the Polycystic Kidney Research Foundation to J.P.C. B.D.C. oncogene expression in 3-week-old cystic kidneys received support from the National Kidney Foundation. suggests that c-fos, c-myc, and c-Ki-ras may be ab- normally regulated. Several possibilities are sug- REFERENCES gested: ( 1 ) that these cystic kidneys are under contin- uous mitogenic stimulation, but passage through the 1 . Grantham JJ: Polycystic kidney disease: A pre- cell cycle is aborted before completion; (2) that some dominance of giant nephnons. Am J Physiol 1 983;244:F3-F 10. step In has escaped normal con- 2. Welling LW, Grantham, JJ: Cystic disease of tnols, giving rise to constitutive expression of the the kidney. In: Brenner BM, Rector FC Jr. eds. proto-oncogenes; or (3) that the elevated pnoto-onco- The Kidney. 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