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-myc, c-ki-ras, renal cysts, 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 cyst 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 gene 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 mitogens (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 genes 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 plasmids 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.