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Kidney International, Vol. 48 (1995), pp. 1451—1458

DNA synthesis is dissociated from the immediate-early response in the post-ischemic kidney

JuDIT MEGYESI, JOHN Di MARl, NoiUDVARHELYI,PETER M. PRICE, and ROBERT SAFIRSTEIN

University of Texas Medical Branch at Galveston, Division of Nephrology, Department of Internal Medicine, Galveston, Texas, USA

DNA synthesis is dissociated from the immediate-early gene response tion of these proteins, the proteins heterodimerize to form a in the post-ischemic kidney. The response of the kidney to ischemic injuly , AP-1, that regulates the expression of other includes increased DNA synthesis, which is preceded by rapid and brief expression of the c-f bs proto-. While the timing of these two often in combination with other proteins [9]. These changes events would suggest that c-Fos participates in an immediate-early gene in gene transcription and post-translational modification of pro- program leading to proliferation, no direct test of this hypothesis exists. teins are observed after growth factor addition and results in the The purpose of these studies was (1) to determine whether c-fos isreplication of the cell in which c-fos is expressed [10]. DNA expressed as part of a typical immediate-early (IE) gene response, which synthesis and completion of the cell cycle usually follow within 12 would require co-expression of c-jun and sensitivity to cycloheximide, and (2) to determine whether the cells expressing c-Fos are the same as those to 24 hours of the expression of the IE genes. While the undergoing DNA synthesis. Northern analysis was performed on renal expression of c-fos and its time course during renal reperfusion mRNA at different times following release of a 50 minute period of renal injury have been well described, other aspects of the IE response hilar clamping. c-jun and c-fos mRNA were rapidly and briefly expressed have not. Neither c-jun nor the sensitivity of the IE response to following renal ischemia and their expression was superinduced by cycloheximide have been examined in detail following reperfusion cycloheximide in a manner typical of an immediate-early gene response. 3H-thymidine autoradiography performed on semi-thin sections from injury of the kidney, and both of these are necessary for the renal intravascularly perfusion fixed kidneys 24 hours following induction of c-fos response to be part of an immediate-early gene cascade [11]. ischemia showed labeled nuclei in cells lining the damaged proximal In many situations, however, cell proliferation is not a conse- tubules of the outer stripe of the Outer medulla, as well as proximal tubules quence of IE and may be followed instead by in the cortex and interstitial cells throughout the kidney. However, immunohistochemical localization of c-Fos and c-Jun protein occurred differentiation [12], a halt in cell cycling [101, or even cell death predominantly in nuclei of the thick ascending limb, distal tubule and [13, 14]. Under these circumstances, IE gene expression is not collecting duct cells. The studies demonstrate that c-fos and c-jun are accompanied by increased DNA synthesis. Thus the function of expressed following renal ischemia as a typical immediate-early gene the renal IE gene response is unclear. The purpose of these response, but they are expressed in cells that do not enter the cell cycle. The failure of the cells to enter the cell cycle may depend on thestudies is to characterize the renal IE response after ischemia co-expression of jun-B and jun-D, which suppress the mitogenic activity of more completely and to determine whether the renal IE response c-Jun in other cells. The data suggest that the IE response following renal occurs in cells that undergo DNA synthesis. Therefore, we ischemia is part of the stress response, which is antiproliferative rather performed Northern analysis, 3H-thymidine incorporation auto- than proliferative. The role of the stress response during renal ischemia and the fate of the cells undergoing it are unknown. radiography and immunocytochemistry to study rats at varying times after release of a 50 minute period of bilateral renal hilar clamping. The results demonstrate that the renal expression of c-jun and The restoration of kidney function following ischemic or neph-c-fos has many of the characteristics of the immediate-early gene rotoxic damage is accompanied by enhanced DNA synthesis andresponse described previously. They also demonstrate that the cell division. Several laboratories have characterized the molecu-predominant cell populations expressing c-jun and c-fos are lar response that accompanies renal repair after ischemic injurydistinct from those undergoing DNA synthesis. The results indi- [1—7]. Prominent among the changes in gene expression observed cate that the JE gene response to ischemia does not serve a is the increased expression of the "immediate-early" gene (IE)proliferative pathway in the cells in which they are expressed and family, including the transcription factor c-fos. c-fos expressionmay be part of a different pathway initiated by stress. alone is insufficient for it to act as a transcription factor. This Methods activity depends on the coexpression of other genes, in particular the members of the Jun family of nuclear proteins [81.Under Animal preparation circumstances that lead to additional post-translational modifica- Ischemia was induced in anesthetized (ketamine, 50 mg/kg) male Sprague-Dawley rats weighing 250 to 500 g by occluding both renal arteries for 50 minutes under sterile conditions. In Received for publication February 25, 1995 sham operated animals, anesthesia, manipulation of the renal and in revised form May 17, 1995 hilus, and exposure of the peritoneal cavity for 50 minutes were Accepted for publication June 19, 1995 performed as in the ischemia studies, but occlusion of the renal © 1995 by the International Society of Nephrology arteries was omitted. Animals were sacrificed and studied at

1451 1452 Megyesi et al: Immediate-early genes and ARF

various times in the reflow period. Cycloheximide at a dose of 1nonimmune species-specific serum, the sections were incubated mg/kg body wt i.p., which has been shown to inhibit proteinwith specific anti-Jun (1:2000; gift of R. Bravo, Bristol-Myers synthesis in rat kidney completely [15], was given two hours before Squibb, Princeton, NJ, USA) or anti-Fos (1:50; Ab-2, rabbit sacrifice to several animals. anti-mouse, Oncogene Science, Uniondale, NY, USA) serum RIVAblotanalysis. Relative levels of mRNA were analyzed byovernight at 4°C. The sections were rinsed in PBS and incubated Northern analysis. Total RNA was isolated from kidneys ofwith biotinylated anti-species immunoglobulin G for 30 minutes at anesthetized animals at varying time periods after renal ischemiaroom temperature before washing in PBS for 20 minutes. The or sham operation by the guanidinium thiocyanate procedure [16]specific immunoreaction was detected with peroxidase-conjugated after intra-arterial perfusion of the kidney with cold 154 mMstreptavidin and 3,3-diamino benzidine and hydrogen peroxide, all NaCl-50 mi tris(hydroxymethyl)aminomethane (Tris) (pH 7.4).according to the manufacturer. Poly(A) RNA was isolated by oligo(dT)-cellulose (type 3; Col- laborative Research, Bedford, MA, USA) chromatography [17]. Autoradiographic studies Poly(A) RNA (5 g/lane) was electrophoresed through formal- Twenty-four hours after reflow, animals (N =2)were injected dehyde-1% agarose gels [18] and transferred overnight to nitro-i.p. with 3H-methyl-thymidine, 1 mCi/kg body wt. After 60 min- cellulose membranes (BA 85, Schleicher and Schuell, Keene, NH,utes, animals were anesthetized and sacrificed. USA) as described [19]. Hybridization was carried out with The kidneys were removed after intravascular perfusion with random primed probes labeled with [32P]deoxycytidine 5'-triphos-freshly prepared 4% paraformaldehyde in PBS. Small tissue phate (dCTP) (500 Ci/mmol) to a sp act of —5X 108 counts/mm blocks (1 x lxi mm) were cut that included tissue of the cortex, (cpm)/JLg DNA [19]. Nitrocellulose blots were prehybridized atthe outer stripe of the outer medulla and the papilla. These blocks 45°C overnight with denatured salmon sperm DNA (0.1 mg/mi) inwere post-perfusion fixed overnight at 4°C in the same buffered 5 >< saline sodium citrate (SSC), 50% deionized formamide, andparaformaldehyde solution and embedded in epon. One micron 0.1% Denhardt's solution [0.1% Ficoll, 0.1% polyvinylpyrrolidine,sections were cut and placed on acid washed glass microscope and 0.1% bovine serum albumin (BSA)] and hybridized at 45°Cslides. The sections were dipped in a 1:1 dilution of Kodak NTB2 overnight in the same solution containing 10% dextran sulfate andemulsion and exposed at 4°C for 1 to 2 weeks. The slides were radiolabeled probe. Nonspecifically bound radioactivity was re-then developed with Kodak D-19 developer, fixed with Kodak moved by extensive washing as described [20]. After washing, the Rapid Fix, washed with water, and stained with methylene-blue blots were air dried and exposed to Kodak XAR film at —70° with and azure II. The percentage of nuclei that were undergoing DNA intensifying screens. Quantitative variabilities of isolation andsynthesis was estimated by the fraction of those nuclei that had transfer of RNAs were accounted for by reprobing the Northernincorporated 3H-thymidine. blots with a cDNA probe for glyceraldehyde-3-phosphate-dehy- drogenase. The sequences used for nick-translation were excised Results with appropriate restriction endonucleases and purified by pre- parative gel electrophoresis. The cDNA for c-fos was a 1 kb c-jun,jun-B, and jun-D were coexpressed with c-fos during renal fragment of v-fos in pfos-1 [21]; murine cDNAS for c-jun (2.6 kbreperfusion injury and cycloheximide superinduced c-fos and c-jun mRNA (Fig. 1) from clone JAC-1), jun-B (1.9 kb fragment in clone 465.20), and Northern analysis of renal mRNA following release of the hilar jun-D (1.7 kb fragment from clone XHJ-12.4) were purchasedclamp revealed rapid and brief expression of c-fos and jun mRNAs from ATCC (Rockville, Maryland, USA), and the cDNA for rat glyceraldehyde-3-phosphate-dehydrogenase was a 1.3 kb frag-in a pattern typical of the immediate-early response (Fig. 1 A, B). An additional characteristic of c-fos and c-jun expression during ment from prGAPDH-13 [221. To quantify levels of fos and jun the IE gene response is their superinduction after cycloheximide mRNA, autoradiograms were scanned in two dimensions (Molec- treatment [23, 24J. Cycloheximide also superinduced c-jun and ular Dynamix Phosphor Image Model 400, Sunnydale, CA, USA)c-fos expression after ischemia (Fig. 1C). Interestingly, cyclohex- and related to the GAPDH signal on the same blot. imide treatment alone increased the expression of these genes, Immunohistochemical localization of Fos and Jun during acute especially c-jun, as has been observed in many cell types [231. The renal failure. Kidneys were removed after intravascular perfusionresults demonstrate the close temporal relationship of renal c-fos with freshly prepared 4% paraformaldehyde in PBS (NaC1 154 m, sodium phosphate, 50 mM, pH 7.3; PBS). Small sagittaland jun expression following ischemia. sections (1 to 2 mm in thickness) were cut and post-perfusion fixed Fos and Jun immunostaining overnight in the same buffered paraformaldehyde solution. After several washes with PBS and a 30 minute period of cryoprotection Minimal c-Fos or c-Jun staining was identified in sections from in 20% sucrose, the tissue was snap frozen in liquid nitrogen. Thinnormal kidney (data not shown). In the ischemic kidney studied (6 to 8 jim) sections were cut in a cryostat and placed onto slidestwo hours after release of the hilar clamp, positive nuclear previously treated with poly-L-lysine to promote better sectionstaining for c-Fos and c-Jun was seen in distal tubule segments of adherence. the cortex (Fig. 2 A, D), thick ascending limbs and collecting ducts The sections were fixed an additional 10 minutes with 4%of the outer medulla (Fig. 2 B, E) and collecting ducts of the inner paraformaldehyde in PBS and rinsed three times with PBS alonemedulla (Fig. 2 C, F). Only occasional staining was observed in before applying the immunoperoxidase procedure to localize Fosproximal tubules within the cortex. The glomeruli were negative. and Jun as described by the manufacturer (Elite ABC Kit, Vector Laboratories, Burlingame, CA, USA). After blocking the endog- Autoradiographic studies enous peroxidase activity with hydrogen peroxide and reducing Ischemic kidney sections were examined for 3H-thymidine nonspecific background antibody staining by incubation withincorporation in nuclear DNA by autoradiography 24 hours thymidine incorpation inwholekidneyDNA atthistimefollowingwhere 14% (N= chosen sincepreviousmeasurements demonstrateahighlevelof following releaseofarterialclamps (Fig.3).Thistimepointwasrenalischemia[5].Silver grainscanbeseenoverlyingnucleiof The effectofcycloheximideinsham operatedanimalsisalsoshown. on theright.C.Northernanalysisof wholekidneymRNA(5tg/lane)onehourfollowingrenalischemia aloneorischemiaandcycloheximideinjection. (5 pg/lane)inshamoperated(control) andischemicanimalsatvaryingtimesfollowingreleaseofthe renal hilarclamp.ThecDNAprobeisindicated Fig. 1. Co-expressionofc-fosandJunduringrenalischemiatheirsuperinduction bycycloheximide.A w

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a.,. . - 4. 4 • Fig. 2. Fos (A—C) and Jun (D—F) immunohistochemist,y of kidney sections from cortex (A and D), outer medulla (B and E), and papilla (C andF) two hours following renal ischemia. Positive immunostaining can be seen in the nuclei of the distal tubules, thick ascending limbs, and collecting ducts (A). Unstained glomeruli (G) and proximal tubules (pt) are also shown. stained nuclei. In the outer stripe of the outer medulla an evenfew labeled nuclei of distal convoluted tubules in the cortex (2%, higher percentage of nuclei (25% of the nuclei of that cell type, NN =2) andin the outer stripe of the outer medulla (2%, N =2). =2)were labeled, especially in cells lining the remnants of theSimilarly, small numbers of labeled nuclei were seen in the proximal tubules which contained necrotic debris. Silver grainspapilla, including interstitial (1.4%) and collecting duct cells were seen overlying nuclei of several interstitial cells, especially in(0.13%). The frequency of labeled nuclei in the control sections the outer stripe of the outer medulla (11%, N =2).There were awas extremely low, data not shown. within thecellsthatexpressthem. cell cycle.Theresultsoftheseexperimentsindicatethat the their expression,and(3)tocorrelatetheexpressionofthesegenes pletely, (2)toidentifythecellswithinkidneyresponsiblefor Silver grainswerenotseenincellsoftheconnectingtubule(CT),thickascendinglimb(Tal)andcollectingduct(CD). necrotic tubulesoftheoutermedulla(NT).Silvergrainscanbefoundoverlyingnucleiinterstitialcells(IS)andpapilla as following renalischemia.Silvergrains(A)canbeseenintheproximaltubules(PT)cortexaswellnucleiiofcellslining Fig. and therapidsynthesis ofc-Fosandc-Junprotein [8],thetwo Given therapidexpressionofc-fos andc-junmRNAafterreflow, nephron identifiesthesecells astheircellularsiteofsynthesis. binding proteinsnotknownto besecreted,localizationindistal the samecelltypesthatexpress c-Fos.Asthesearenuclear emia. Inaddition,wenowshow thatc-Junproteinisexpressedby inantly inthedistalnephronfollowing abrieferperiodofisch- Brown andSchwarz[25],whoalsoshowc-fosexpressionpredom- nephron. OurfindingsareinagreementwiththoseofWitzgall, almost exclusivelyrestrictedtothenucleiofcellsdistal response. Immunostainingforc-Fostwohoursafterreflow was c-jun responseafterrenalreperfusioninjuryasatypical IE their superinductionaftercycloheximideestablishthec-fos and lowing renalischemiaotherthanthesupportofDNAsynthesis immediate-early generesponseservessomeotherfunction fol- to atleastoneknownoutcomeoftheirexpression,entryintothe immediate-early generesponseinrenalischemiamorecom- The rapidandbriefchangesinc-junc-fosexpression The purposeofthesestudieswas(1)tocharacterizethe 3. DNAsynthesisinthekidneyafterrenalischemia.Photomicrographsofcortex(A),outermedulla(B),andpapilla(C) ratkidney24

Discussion Ii

4 Megyesi etal:Immediate-earlygenesandARF by UVirradiationandoxidant stress[27].Thesestimuliinduce growth factorsactivatethisprogram. Ofparticularinterestinthe them toentryintothecellcycle. Otherstimuliinadditionto the immediate-earlygeneprogram inthesecellsdoesnotcommit including Egi--1[26]andJE[6],establishthattheactivation of that areexpressedinthedistalnephronfollowingrenalischemia, their prominentlabelingbyc-Fosandc-Junantibodies. duct cellsshowednuclearlabeling.Thisisinmarkedcontrast to limb andonlyaveryfewdistalconvolutedtubuleorcollecting medulla. Nolabeledcellsweredetectedinthethickascending interstitial spacesurroundingtubulesofthecortexand outer replication. Therewasalsoprominentlabelingofcellsin the suggests thatanevengreaterpercentageofcellswereundergoing of outerstripecellslabeledbybolusinjection3H-thymidine the necroticproximalstraighttubules.Thehighpercentage(25%) tubules andincellsliningthelumensofpresumedremnants of DNAsynthesiswereidentifiedincorticalproximalconvoluted DNA occurredinadifferentpopulationofcells,Prominentsites expression. hour periodshouldberepresentativeoftheprotein transcriptional events verysimilarlytothose initiatedbygrowth setting ofischemiaistheactivation oftheimmediate-earlygenes These studiesaddtothenumberofimmediate-earlygenes In contrast,theincorporationof3H-thymidineintonuclear A tr. • ci

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Fig. 3. Continued Megyesi et al: Immediate-early genes and ARF 1457 factors, including a typical IE gene response, but do not provoke mal growth factor mRNA and decreased EGF excretion in ARF. proliferation. Instead, this response is usually antiproliferative. Kidney mt 36:810—815, 1989 The rapid and coordinate expression of jun-B and jun-D induced 5. SAFIRSTEIN R, PRICE PM, SAGGI SJ, HARRIS RC: Changes in gene expression after temporary renal ischemia. Kidney mt 37:1515—1521, by renal ischemia is also observed following oxidative and ionizing 1990 radiation-induced cell stress [28]. These members of the Jun 6. SAFIRSTEIN R, MEGYESIJ,SAGGI SJ, PRICE PM, PooN M, ROLLINS BJ, family negatively regulate the mitogenic and transformation ac- TAUBMAN MB: Expression of cytokine-like genes JE and KC is tivities of c-Jun in most circumstances. Thus Jun-D negatively increased during renal ischemia. Am J Physiol 261:F1095—F1 101, 1991 7. SAFIRSTEIN R, MEGYESI J, SAGGI SJ, HOYER JR, PRICE PM: Molecular regulates fibroblast growth and antagonizes transformation by ras and cellular responses during renal ischemia. (abstract) JASN 3:714, [29]. Jun-B inhibits both the transforming and transactivation 1992 activity of c-Jun in fibroblasts [30]. Another example of the 8. ANGEL P, KARIN M: The role of Jun, Fos and the AP-1 complex in apparent antagonism of the proliferative affects of c-Jun by Jun-B cell-proliferation and transformation. Biochim Biophys Acta 1072: of is the potentiation of NGF-induced proliferation of PC12 cells 129—157, 1991 9. Cuju R, BOYLE WJ, MEEK J, SMEAI T, HUNTER T, KARIN M: The with antisense Jun-B DNA [311.ThusJun-B and Jun-D may act as c-Fos protein interacts with c-Jun/AP-1 to stimulate transcription of modulators of the mitogenic activity of c-Jun in the distal nephron AP-1 responsive genes. Cell 54:541—552, 1988 and suppress its mitogenic potential. 10. DAvis Ri: The mitogen-activated protein kinase signal transduction The transcriptional activation of c-jun during the IE response pathway. J Biol Chem 268:14553—14556, 1993 11. BRAVO R: Genes induced during the GOIG1 transition in mouse requires the phosphorylation of pre-existing c-Jun at specific fibroblasts. Cancer Biol 1:37—46, 1990 serine and threonine residues in its N-terminal transactivating12. MULLER R, WAGNER EF: Differentiation of F9 teratocarcinoma stem domain by proline directed serine-threonine kinases [10, 32, 331. cells after transfer of c-fos proto-. Nature 311:438—442, Theprincipal c-Jun kinases activated by stress that have been 1984 identified to date are the p54 and JNK1 kinases, also known as the 13. SMEYNE RJ, VENDRELL M, HAYWARDM,BAKER SJ, MIAO GG, SCHILLINO K, ROBERTSON LM, CURRAN T, MORGAN JI: Continuous stress-activated protein kinases, or SAPK [341.Theexpression of c-fos expression precedes programmed cell death in vivo. Nature the IE genes in cells not undergoing proliferation and not 363:166—169, 1993 prominently damaged morphologically by ischemia suggests that14. GOLDSTONE SD, LAVIN MF: Prolonged expression of c-jun and the increase in c-fos and c-jun expression could occur as part of associated activity of the transcription factor AP-1, during apoptosis in a human leukaemic cell line. Oncogene 9:2305—2311, 1994 this stress response [27, 34]. The induction of c-jun by cyclohex- 15. LEVINE BS, Ho LD, PASIECZNIK K, COBURN JW: Renal adaptation to imide in normal animals is consistent with the constitutive expres- phosphorus deprivation: Characterization of early events. I Bone sion of the p54 SAPK (Fig. 1C) in the kidney as this SAPK is Miner Res 1:33—40, 1986 activated by cycloheximide [35]. The test of this hypothesis will 16. 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