Expression of cytokine-like during renal ischemia
genes JE and KC is increased
ROBERT SAFIRSTEIN, JUDITH MEGYESI, SUBODH J. SAGGI, PETER M. PRICE, MICHAEL POON, BARRETT J. ROLLINS, AND MARK B. TAUBMAN Department of Medicine, Renal, Cardiology and Molecular Medicine Divisions, Department of Biochemistry, and Brookdale Center for Molecular Biology, Mount Sinai School of Medicine, and Division of Medicine, Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115
SAFIRSTEIN, ROBERT, JUDITH MEGYESI, SUBODH J. SAGGI, PETER M. PRICE, MICHAEL POON, BARRETT J. ROLLINS, AND MARK B. TAUBMAN. Expression of cytokine-like genes JE and KC is increased during renal &hernia. Am. J. Physiol. 261
(Renal Fluid Electrolyte Physiol. 30): F1095-FllOl, 1991.Both mitogenic and inflammatory phenomenaaccompany the renal responseto ischemicinjury. Previous studieshave shown that several nuclear-binding membersof the immediate early genesare prominently expressedafter renal ischemiaand may underlie the mitogenic responseto such injury. We now report on the expressionof JE and KC, other growth-factor-responsive genesthat codefor small secretedglycoproteins with cytokinelike properties, which may play a role in inflammation. The expressionof the immediateearly genesJE and KC was determined in rat kidney tissue at varying time points after release of a 50-min period of bilateral renal hilar clamping. Relative levelsof mRNA for JE and KC were analyzed by Northern blot analysis of cortical and outer stripe mRNA. KC mRNA rose rapidly to peak values at 1 h and returned toward low baseline levels by 24 h after releaseof the hilar clamp. By contrast, JE mRNA reachedpeak levels later and remained elevated for at least 96 h after ischemia.JE antigen was localized immunocytochemically to the apical regionsof the cortical and medullary thick ascendinglimbs as well as in the lumen of the distal nephron in ischemic kidneys. Cells of the glomerulus and proximal tubules were negative for JE antigen. In contrast to the increasein JE and KC mRNA, steady-state levels of uromodulin (Tamm Horsfall) mRNA, a cytokine binding protein also made by the thick ascending limb, declined to virtually undetectable levels by 24 h after ischemia.Thus the increases in JE and KC are not generalized phenomena.These studies demonstratethat the kidney rapidly expressesgenesencoding leukocyte chemoattractants in responseto ischemia. JE and KC may serve the renal regenerative responseeither by promoting the movement of cells along the basementmembrane of injured tubules or by attracting leukocytes into the ischemic kidney. The decline of the cytokine binding Tamm-Horsfall protein may also increasethe activity of locally generated or filtered cytokines within the kidney. ischemic renal failure; immediate early genes JE and KC; Tamm-Horsfall; cytokines and regeneration
and nephrotoxic acute renal failure requires replacement of dead tubular cells with living ones that restore the continuity of the renal epithelium (30). During this process normally quiescent renal cells increase their nucleic acid synthesis and RECOVERY FROM ISCHEMIC
0363-6127/91
$1.50
undergo mitosis (30). These phenomena occur in most forms of acute renal injury, suggesting a similar underlying mechanism. Entry into the cell cycle is largely coordinated by changes in gene expression. Growth-promoting signals reach the nucleus by a variety of intracellular pathways and induce the transcription of many genes (29). First among them are the so-called immediate early genes, whose increased expression begins almost immediately after such stimulation, lasts only briefly, and does not depend on new protein synthesis (1). Some of these immediate early genes code for nuclear or DNA binding proteins that presumably initiate a genetic cascade ending in cell division. The transcription of these genes is increased by ischemic renal damage in a manner that is strikingly similar to that induced by growth factor stimulation of quiescent cells in culture. The prototype of the nuclear binding members of the immediate early genes, c-fos mRNA, increases almost immediately after release of renal hilar clamping (19, 26). Its induction is short lived and precedes the peak of renal DNA synthesis. A similar increase is also observed for the immediate early gene Egr 1 (19, 26). It is highly likely, therefore, that the immediate early genes somehow serve the renal regenerative response to ischemia. Renal injury also has profound effects on the production of epidermal growth factor (EGF). Ischemia and cisplatin reduce preproEGF mRNA and reduce EGF excretion (26, 27) but, in contrast to the changes in the immediate early genes, preproEGF mRNA levels fall more gradually and remain low for a prolonged period of time. The renal failure-induced reduction of preproEGF mRNA is of interest, since it cannot be ascribed to the simple loss of the cells that produce it and because the thick ascending limb and distal tubule, which are its only sites of synthesis, undergo morphological changes that are mild, reversible, and transient after each of these insults (6). The movement of regenerating cells along the basement membrane of injured tubules (30) and the accumulation of leukocytes into the kidney (17) are important responses to renal ischemia. Although their numbers are small, recent studies on the role of leukocytes during renal ischemia (13, 17) show them to be important determinants of the functional and morphological manifes-
Copyright 0 1991 the American Physiological Society
F1095
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on September 7, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
F1096
JE AND
KC
EXPRESSION
tations of ischemic renal failure. Possible mediators of these events are unknown. The genes JE and KC, originally identified as immediate early genes induced by platelet-derived growth factor in mouse 3T3 cells, appear to code for cytokines more involved in inflammation rather than growth. Sequence and expression analysis shows that unlike other early response genes, such as cfos, JE and KC encode secreted glycoproteins (18, 23). The only known function of the JE and KC products is chemotaxis. The KC protein is chemotactic for neutrophils (31) and is a member of a superfamily of proteins that includes platelet factor 4 and connective tissueactivating peptide III. JE is chemotactic for monocytes (32) and smooth muscle cells (15) and is the rat homologue of the human monocyte-specific chemotactic factor MCP-1 (32). These observations suggest that the JE and KC products are linked in some fashion to wound healing and inflammatory responses (31). Increased expression of these genes has also recently been observed after vessel wall injury (15), where it is speculated that they may play an important role in the inflammatory response to such injury in vivo. To determine whether the expression of KC and JE was increased by renal ischemia, relative levels of their mRNAs were determined by Northern analysis at varying times after a 50-min period of renal hilar clamping. Because of the cytokine characteristics of these proteins, we also examined the expression of the renal cytokine binding Tamm-Horsfall (TH) protein, whose cDNA structure and renal site of synthesis are similar to preproEGF (9). The results show that the expression of JE and KC increases and TH decreases after renal ischemia. Immunocytochemical studies show that JE product is localized to the thick ascending limb and distal tubule, the same sites of EGF and TH production and demonstrate that the thick ascending limb is a prominent site of cellular reaction to renal ischemic damage. METHODS
DURING
RENAL
ISCHEMIA
Poly(A)+ RNA was isolated by oligo(dT)-cellulose (type 3; Collaborative Research, Bedford, MA) chromatography (18). Poly(A)’ RNA (5 and 7.5 pg/lane) was electrophoresed through formaldehyde-l% agarose gels (12) and transferred overnight to nitrocellulose membranes (BA 85, Schleicher and Schuell, Keene, NH) as described (14). Hybridization was carried out with nick-translated DNAs labeled with [32P]deoxycytidine 5’-triphosphate (dCTP) (500 Ci/mmol) to a sp act of - 5 x lo8 counts/ min (cpm)/pg DNA (22). Nitrocellulose blots were prehybridized at 45°C overnight with denatured salmon sperm DNA (0.1 mg/ml) in 5 X saline sodium citrate (SSC), 50% deionized formamide, and 0.02% Denhardt’s solution [ 0.02% Ficoll, 0.02% polyvinylpyrrolidine, and 0.02% bovine serum albumin (BSA)] and hybridized at 45°C overnight in the same solution containing 10% dextran sulfate and radiolabeled probe. Nonspecifically bound radioactivity was removed by extensive washing as described (33). After washing, the blots were air dried and exposed overnight to Kodak XAR film at -70” with intensifying screens. Quantitative variabilities of isolation and transfer of RNAs were accounted for by reprobing the Northern blots with a cDNA probe for glyceraldehyde-3-phosphate-dehydrogenase. The sequences used for nick translation were excised with appropriate restriction endonucleases and purified by preparative gel electrophoresis. The cDNAs for JE [a 750-base pair (bp) Pst I fragment of pBC-JE]; KC (a 820-bp Pst I fragment of pKC); TH (a 2.2-kb Eco RI fragment in pBR322 (20); murine preproEGF (a 700-bp Pst I fragment of pmegfl0 (21)); and rat glyceraldehyde-3-phosphate-dehydrogenase from prGAPDH-13 (7) were gifts of the authors. To quantify levels of JE, KC and TH mRNA, autoradiograms were scanned in two dimensions using an Apple scanner and densitometry employing 256 gray scales on an Apple Macintosh IIcx computer using Image 1.36 software (Wayne Rasband, National Institutes of Health) and related to the strength of the GAPDH signal on the same blot.
General Methods
Immunocytochemical localization of JE during acute renal failure. Kidneys were removed at varying times
Animal preparation. Male Sprague-Dawley rats weighing 250-500 g were fed a standard rat chow (Rat Chow 5012; Ralston Purina, St. Louis MO, containing 22.8% protein, 4.5% fat, and 4.6% fiber and water) and offered tap water ad libitum. After a period of 4-6 days on this diet, rats were anesthetized (ketamine, 75 mg/kg), and both renal arteries were occluded for 50 min under sterile conditions. In sham-operated animals, anesthesia, manipulation of the renal hila, and exposure of the peritoneal cavity for 50 min were performed as in the ischemia studies, but occlusion of the renal arteries was omitted. Animals were killed at various times in the reflow period as required by the specific protocols. RNA blot analysis. Relative levels of mRNA were analyzed by Northern and dot-blot analysis. Total RNA was isolated from the cortex and outer stripe of the outer medulla from anesthetized animals at varying time periods after renal ischemia or sham operation by the guanidinium thiocyanate procedure (5) after intra-arterial perfusion of the kidney with cold 154 mM NaCl-50 mM tris(hydroxymethyl)aminomethane (Tris) (pH 7.4).
after release of the renal hilar clamp and after intravascular perfusion with freshly prepared 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Small sagittal sections (l-2 mm in thickness) were cut and postperfusion fixed for no longer than 30 min in the same buffered paraformaldehyde solution. After several washes with phosphate-buffered saline (PBS) (NaC1154 mM, sodium phosphate, 100 mM, pH 7.3) and a 30-min period of cryoprotection in 20% sucrose, the tissue was snap frozen in liquid nitrogen. Thin (6-8 pm) sections were cut in a microtome and placed onto slides previously treated with poly-L-lysine to promote better section adherence. Sections were stored at -70°C before proceeding to the immunocytochemical steps. On removal from storage, the sections were fixed an additional 10 min with 4% paraformaldehyde in 0.5 M Tris (pH 7.6) and rinsed 3 times in 0.5 M Tris buffer. The sections were then treated with 0.3% hydrogen peroxide in methanol for 20 min to reduce nonspecific background staining by endogenous peroxidase activity and rinsed three times in 0.5 M Tris. To block nonspe-
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on September 7, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
I_
.
.
*-
JJ!S ANLJ
*.,-.
-vv.-.---AI-.
KU
.
DURING
RENAL
F1097
ISCHEMIA
k!Xk'lt.l~LZS5lUN
TABLE
1. Effect of ischemia on renal KC and JE mRNA
No. of h after Control 1 4 24 48 98 168
KC
n
JE
n
23+13 127f31* 75*13t 45k12 ND 11*15 ND
4 4 4 4
19+s 43*15 56+6$ 73-t23t Sl+S§ 69+6§ 41+14
6 3 6 6 3 3 2
ischemia
2
Values are means f SE; n = no. of determinations. Densitometric readings for KC and JE mRNA were divided by GAPDH readings and are in percent. ND, not determined. *P < 0.025; t P < 0.05, $ P < 0.005, and $ P < 0.001, significantly different from control.
FIG. 1. Northern analysis of KC mRNA. Poly(A)+ RNA (5 pg/lane) isolated from male Sprague-Dawley rat kidney cortex was applied to each lane from control (1st lane) and ischemic animals (Zones 2-4) at times indicated. Location of 18s RNA is shown, Z& here and in Figs. 2-5. KC (top) and GAPDH (bottom) cDNA probes were used as described in METHODS.
TH GAPDH FIG. 4. Northern analysis of TH mRNA. Poly(A)+ RNA (5 pg/lane) was applied to each lane from control (1st lane) and ischemic animals (lanes 2-6) at times indicated. TH and GAPDH cDNA probes were used as described in METHODS.
FIG. 2. Northern analysis of JE mRNA. Poly(A)+ RNA (7.5 rg/ lane) was applied to each lane from control (1st lane) and ischemic animals (.&es 2-6) at times indicated. JE and GAPDH cDNA probes were used as described in METHODS.
--*-- KC !
-
JE
:
0
24
48
Time
72
96
120
144
166
(hrs)
FIG. 3. Relative levels of JE and KC mRNA in ischemic kidneys. JE and KC mRNA levels were determined by 2-dimensional densitometry of scanned audioradiograms. Levels were normalized to those of GAPDH mRNA and are expressed relative to control nonischemic kidney levels where control = 1. Each point represents mean of at least 3 animals.
with a specific anti-JE serum at 1:200 dilution in TGBA containing the same concentration of goat serum and biotin overnight at 4°C. The anti-JE serum was raised in New Zealand White rabbits against mouse JE by methods previously described (24). The slides were then rinsed in 0.5 M Tris and incubated with biotinolated anti-rabbit immunoglobulin G (IgG) (Amersham) for 1 h at room temperature. The slides were then rinsed in 0.5 M Tris three times and incubated further in peroxidase-conjugated streptavidin (Jackson Immunoresearch Labs, West Grove, PA) diluted 1:lOO for 1 h at room temperature. After slides were rinsed in 0.5 M Tris the reaction was developed in 0.05% 3,3’diaminobenzidine and 0.01% hydrogen peroxide in 0.5 M Tris for 5 min and followed by a brief wash in 0.5 M Tris and water and dehydrated in graded alcohols and xylene. After counterstaining with hematoxylin, the slides were mounted with a cover slip and observed under a light microscope. The intensity of the immunohistochemical staining was scored using the following criteria: 0 = no detectable localization; + = weak, ++ = moderate and +++ = strong staining. Sections from control animals, as well as substitution of the primary antisera with nonimmune serum, were used as negative controls. RESULTS
Figure 1 is a representative Northern blot anlysis of from normal and ischemic animals. KCspecific mRNA was present at very low levels in normal kidneys. Levels of KC mRNA were markedly induced by renal &hernia, peaking between 1 and 4 h. Levels returned to baseline within 24 h and remained low for up to 1 wk after ischemia. This rapid induction and fall of KC mRNA
cific antibody binding sites the sections were incubated with nonimmune goat serum (20%) and biotin (0.1%) in Tris-gelatin-bovine albumin (TGBA) buffer (gelatin 0.1%; BSA 0.1%; NaN3 0.1% in 0.5 M Tris (pH 7.6) for 1 h at room temperature. Then the section was incubated
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on September 7, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on September 7, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
JE
AND
KC
EXPRESSION
KC mRNA conforms to the general characteristics of immediate early gene expression (1). Figure 2 shows results of Northern blot analysis of JE mRNA. JE mRNA was also present at low levels in control kidneys. Like KC, JE mRNA was rapidly induced following renal ischemia. However, unlike KC, elevated levels of edE mRNA persisted for up to 1 wk, peaking at between 48-96 h. Figure 3 is graphic representation of densitometric analysis of KC and JE mRNA levels pooled from multiple experiments (Table 1). Levels of JE and KC were normalized to GAPDH and are expressed as arbitrary units relative to a control of one for nonischemic kidneys. Each time point is an average of at least three individual animals. KC mRNA peaked at 4.8 t 1.8fold above control at 1 h (n = 3, P ~0.025). JE mRNA increased to 2.9 & O&fold above control (n = 6, P CO.005) at 4 h and peaked at 4.3 & 0.6 fold (n = 3, P CO.001) above control levels at 48 h after release of the hilar clamp. Figure 4 shows representative Northern blot analysis of 2X mRNA. In contrast to JE and KC mRNA, TH mRNA is high in normal kidney. Beginning at -6 h (data not shown), TH mRNA levels fell progressively to only 21% of control at 24 h and 10% of control at 48 h. TH mRNA levels were still greatly reduced at 96 h (21% of control values) but returned to control levels 1 wk after ischemia. In cell culture, JE protein is rapidly secreted after its mRNA is induced (23, and unpublished observations). In addition, JE protein is present in a variety of cells including fibroblasts (24)) vascular smooth muscle (15)) and endothelial cells (25). Thus it was of interest to determine the precise localization of the JE product in the kidney. Using an anti-JE antiserum that identifies a single protein on Western blots (24), we performed immunocytochemical studies of renal tissue at early time points following the induction of JE. The results of these immunocytochemical studies of JE protein localization are shown in Fig. 5. Kidney sections of the outer stripe of the outer medulla in sham-operated control (A and C) and in 4-h postischemic animals (B and D) are shown. In both unstained and stained sections of the kidney (B and D) JE protein, indicated by the arrows, was seen at the luminal aspect of the thick ascending limbs of Henle’s loop in the ischemic kidneys and was not detectable in the control kidneys (A and C). No reaction product was detected in glomeruli or proximal tubules. Treatment of slides with 0.3% hydrogen peroxide to reduce endogenous peroxidase activity and nonimmune goat serum to block nonspecific antibody binding sites was used to reduce background activity. Incubation with nonimmune rabbit serum did not reveal specific staining. DISCUSSION
The present studies show that during the early reflow period of renal ischemia, the expression of JE and KC,
DURING
RENAL
ISCHEMIA
F1099
two immediate early genes induced by growth factor and lipopolysaccharide addition to cells in culture, is increased. But unlike other immediate early genes such as c-fos and Egr-1, whose expression is also increased following renal ischemia, the increased expression of JE was prolonged and peaked at least 4 days after ischemia. Such prolonged expression of JE by the ischemia-damaged kidney is unique. Mitogenic (18, 23) and lipopolysaccharide-stimulated (10) expression of JE in cell culture is short lived. In addition, we have recently reported that balloon injury of rat and rabbit aorta (15) induced a transient rise in JE mRNA levels. This suggests that the mechanism for induction and regulation of JE mRNA in the kidney may be different from that in balloon-injured vessels and distinct from those previously examined in cell culture. In addition the elevated levels of JE mRNA persisted well beyond the ischemiainduced peak of DNA synthesis that occurs at 24 h after induction of ischemia (26). These observations coupled with the localization of JE to the thick ascending limb, a site not prominent for cell division after ischemia (6), would indicate that JE and probably KC serve other roles in renal ischemia not specifically linked to cell division. What these roles are can only be speculated on at the present time, but the chemotactic and inflammatory properties of JE and KC would suggest that they may promote the movement of regenerating cells to restore the integrity of the tubule epithelium or promote the accumulation of leukocytes into the ischemic kidney (17). The coincidental fall in TH expression after ischemia would also enhance the activity of locally produced or filtered cytokines, if, as suggested, it functions as a cytokine-binding and inactivating protein (20). Thus the reciprocal changes in JE, KC, and TH may modulate the inflammatory response to renal ischemia. Such enhancement in cytokine activity would also be expected to have effects on renal function (28). JE antigen was immunocytochemically localized to the thick ascending limb and distal tubule of ischemic kidneys. It is important to note that finding the JE protein on the luminal surface of the thick ascending limb does not necessarily indicate that these cells produce it. JE may be made elsewhere and bind to sites on these cells. However, in the absence of significant JE antigen elsewhere in the kidney, the thick ascending limb should be considered the most likely site of JE production. Previous studies have established that the thick ascending limb undergoes prominent changes in gene expression during renal ischemia, both positive and negative. Epidermal growth factor mRNA, made only by the thick ascending limb and distal tubule of normal kidneys (24) falls markedly during renal ischemia (26), whereas the product of Egr-1, a nuclear binding protein that increases after renal ischemia, is also found in the nuclei of the thick ascending limb (3). The present studies demonstrate that ischemia induces a fall in levels of TH mRNA, also produced by the thick ascending limb (5). The pathophysiological
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on September 7, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
FllOO
JE
AND
KC
EXPRESSION
DURING
significance of these changes in thick ascending limb transcriptional activity is unknown, but recent studies on the isolated perfused kidney have focused attention on the possible role of the thick ascending limb in the pathogenesis of acute renal failure (4). The consequences of the altered expression of genes by the thick ascending limb remains to be established, but defects in salt transport have been documented in apparently morphologically intact thick ascending limbs studied in situ (16) or by perfusion of isolated segments of thick ascending limbs of ischemic animals (8). The thick ascending limb may be a crucial element in the renal response to ischemia as these rapid and long lasting changes in gene expression would indicate. Studies designed to identify how these genes are regulated and how to alter their expression should provide important clues to their functional significance and lead to novel strategies designed to maximize recovery from renal failure. We thank Dr. George Acs for his helpful suggestions during the course of these studies. This work was supported in part by a grant from the National Kidney Foundation of New York-New Jersey to S. Saggi, National Institutes of Health Grants HL-43302 and CA-53901, and a grant-inaid from the American Heart Association, New York Affiliate to M. Taubman. This is publication No. 48 from the Brookdale Center for Molecular Biology. Address for reprint requests: R. Safirstein, Dept. of Medicine, Renal Division, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029 Received 12 December 1990; accepted in final form 24 July 1991. REFERENCES H. MCDONALD-BRAVO, J. Complexity of the early genetic response to growth factors in mouse fibroblasts. 1Mol. Cell. Biol. 8: 2140-2148, 1988. 2. AVIV, H., AND P. LEDER. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc. Nutl. Acud. Sci. USA 69: 1408-1412, 1972. 1. ALMENDRAL, BURCKHARDT,
J. M., D. SOMMER, J.PERERA,
AND R. BRAVO.
3. BONVENTRE, J. V., A. J. OUELLETTE, BROWN. Localization of the EGR-1
V. P. SUKHATME,
AND D.
protein product to the distal nephron after renal ischemia and reperfusion (Abstract). CZin. Res.
38: 442A, 1990. 4. BREZIS, M., S. ROSEN,
F. H. EPSTEIN. Selective vulnerability of the medullary thick ascending limb to anoxia in the isolated perfused rat kidney. J. CZin. Inuest. 73: 182-190, 1983. 5. CHIRGWIN, J. M., A. E. PRZYBYLA, R. J. MACDONALD, AND W. J. RUTTER. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294-5299,1979. P. SILVA,
AND
6. DONOHOE, J. F., M. A. VENKATACHALAM, N. G. LEVINSKY. Tubular leakage and
ischemia: structural-functional
D. B. BERNARD,
AND
obstruction after renal correlations. Kidney Int. 13: 208-
222,1978. 7. FORT, P., L. MARTY, P. JEANTEUR, AND
M. PIECHACZYK, S. E. SABROUTY, C. DANI, J. M. BLANCHARD. Various rat adult tissue express only one major mRNA species from glyceraldehyde-3phosphate-dehydrogenase multigenic family. NucZ. Acid Res. 13:
1431-1442,1985. 8. HANLEY, M.
J. Isolated nephron segments in a rabbit model of ischemic acute renal failure. Am. J. Physiol. 239 (Renal Fluid Electrolyte Physiol. 8): F17-F23, 1980. 9. HESSION, C., J. M. DECKER, A. P. SHERBLOM, S. KUMAR, C. C. YUE, R. J. MATTALIANO, R. TIGARD. E. KAWASHIMA, U. SCHMEISSNER, S. HELETKY ET AL. Uromodulin (Tamm-Horsfall glycoprotein): A renal ligand for lymphokines. Science Wash. DC 237: 1479-1484,1987. 10. INTRONA, M., R. C. BART, ILTON, AND D. 0. ADAMS.
JR., C. S. TANNENBAUM, T. A. HAMThe effect of LPS on exnression of the
RENAL
ISCHEMIA
early “competence” genes JE and KC in murine peritoneal macrophages. J. Immunol. 138: 3891-3896, 1987. 11. KLAUSNER, J. M., I. S. PATERSON, G. GOLDMAN, L. KOBZIK, C. RODZEN, R. LAWRENCE, C. R. VALERI, D. SHEPRO, AND H. B. HECHTMAN. Postischemic renal injury is mediated by neutrophils and leukotrienes. Am. J. Physiol. 256 (Renal Fluid Electrolyte Physiol. 25): F794-F802, 1989. 12. LEHRACH, H., D. DIAMOND, J.
M. WOZNEY, AND H. BOEDTKER. RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry 16:
4743-4751,1977. 13. LINAS, S. L., J. E. REPINE.
P. F. SHANLEY, D. WHITTENBURG, E. BERGER, AND Neutrophils accentuate ischemia reperfusion injury in isolated rat kidneys. Am. J. Physiol. 255 (Renal Fluid Electrolyte
Physiol. 24): F728-F735, 1985. 14. MANIATIS, G. E., F. FRITSCH, AND J. SAMBROOK. Molecular CZoning Manual. New York: Cold Spring Harbor, 1982, p. 383-386. 15. MARMUR, J. D., V. 1;. FRIEDRICH, JR., M. ROSSIKHINA, B. J. ROLLINS, AND M. B. TAUBMAN. The JE gene encodes smooth
muscle chemotactic factor that is induced by vascular injury (Abstract). Circulation 82: 698, 1990. 16. MASON, J., H.-U. GUTSCHE, L. MOORE, AND R. MULLER-SUUR. The early phase of experimental acute renal failure IV. The diluting ability of the short loops of Henle. Pfluegers Arch. 279: 11-18, 1979. 17. OLOF, P., A. HELLBERG, 0. T. KALLSKOG, G. OJTEG, AND M. WOLGAST. Peritubular capillary permeability and intravascular RBC aggregation after ischemia: effects of neutrophils. Am. J. Physiol. 258 (Renal Fluid Electrolyte Physiol. 27): FlO18-F1025, 1990. 18. OQUENDO, P., J. ALBERTA, W. DUANZHI, J. C. GRAYCAR, R. DERYNCK, AND C. D. STILES. The platelet- derived growth factorinducible KC gene encodes a secretory protein related to platelet a-granule proteins. J. BioZ. Chem. 264: 4133-4137, 1989. 19. OUELLETTE, BONVENTRE.
A. J., R. A. MALT,
V. P. SUKHATME,
AND J. V.
Expression of two “immediate early genes, Egr-1 and c-fos, in response to renal ischemia and during renal hypertrophy in mice. J. CZin. Invest. 85: 766-771, 1990. PENNICA, D., W. J. KOHR, W.-J., KUANG, D. GLAISTER, B. B. AGGARWAL, E. CHEN, AND D. V. GOEDDEL. Identification of human uromodulin as the Tamm-Horsfall urinary glycoprotein.
21 Science Wash. DC 236: 83-88, 1987. . RALL, L. B., J. SCOTT, B. I. BELL, R. J. CRAWFORD, J. D. PENSCHOW, H. D. NIALL, AND J. P. COGHLAN. Mouse prepro-epiderma1 growth factor synthesis by the kidney and other tissues. Nature Lord. 313: 228-231,1985. 22* RIGBY, P. W. J., M. DIECKMAN,
C. RHODES, AND P. I. BERG. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. BioZ. 113: 237251,1977.
AND C. D. STILES. Cloning and 23* ROLLINS, B. J., E. D. MORRISON, expression of JE, a gene inducible by platelet-derived growth factor and whose product has cytokine-like properties. Biochemistry 85: 3738-3742,1988.
24* ROLLINS, B. J., P. STIER, T. ERNST, AND G. G. WONG. The human homolog of the JE gene encodes a monocyte secretory protein. MOL. CeZZ. BioZ. 9: 4687-4695,
1989.
E. J. LEONARD, AND J. S. POBER. 25. ROLLINS B. J., T. YOSHIMURA, Cytokine-activated human endothelial cells synthesize and secrete a monocyte chemotractant, MCP-l/JE. Am. J. Path. 136: 12291233,199O.
R., P. M. PRICE, S. J. SAGGI, AND R. C. HARRIS. 26. SAFIRSTEIN, Changes in gene expression after temporary renal ischemia. Kidney Int. 37: 1515-1521,199O. 27. SAFIRSTEIN, R., A. ZELENT,
mRNA and diminished Kidney Int. 28. SCHREINER,
AND P. M. PRICE. Reduced preproEGF EGF excretion during acute renal failure.
36: 810-815,1989. G. F., AND D. E. KOHAN.
Regulation of renal transport and hemodynamics by macrophages and lymphocytes. Am. J. Physiol. 258 (Renal
FZuid
Electrolyte
Physiol.
27): F761-F767,
1990.
29. STILES C. D. The biologic role of oncogenes- insights from plateletderived growth factor. Cancer Res. 45: 5215-5218,1985. 30. TOBACK, F. G. Control of renal regeneration after acute tubule necrosis. In: NephroZogy, edited by R. R. Robinson. New York: &ringer-Verlae. 1984. D. 748-762.
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on September 7, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
JE AND KC EXPRESSION 31. WATANABE,
K., K. KONISHI, M. FUJIOKA, S. KINOSHITA, AND H. The neutrophil chemoattractant produced by the rat kidney epithelioid cell line NRK-52E is a protein related to the KC/gro protein. J. Biol. Chem. 264: 19559-19563, 1989. 32. YOSHIMURA, T., AND E. J. LEONARD. Secretion by human fibroblasts of monocyte chemoattractant protein-l, the product of gene NAKAGAWA.
DURING
RENAL
ISCHEMIA
FllOl
JE. J. Immunol. 144: 2377-2383,199O. 33. ZELENT, A. Z., M. A. SELLS, P. M. PRICE, A. MOHAMAD, G. Acs, AND J. K. CHRISTMAN. Murine cells carrying integrated genomes of hepatitis B virus DNA transcribe RNA from endogenous promoters on both viral strands and express middle and major viral envelope proteins. J. Viral. 61: 1108-1115, 1987.
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on September 7, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
genes JE and KC is increased
ROBERT SAFIRSTEIN, JUDITH MEGYESI, SUBODH J. SAGGI, PETER M. PRICE, MICHAEL POON, BARRETT J. ROLLINS, AND MARK B. TAUBMAN Department of Medicine, Renal, Cardiology and Molecular Medicine Divisions, Department of Biochemistry, and Brookdale Center for Molecular Biology, Mount Sinai School of Medicine, and Division of Medicine, Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115
SAFIRSTEIN, ROBERT, JUDITH MEGYESI, SUBODH J. SAGGI, PETER M. PRICE, MICHAEL POON, BARRETT J. ROLLINS, AND MARK B. TAUBMAN. Expression of cytokine-like genes JE and KC is increased during renal &hernia. Am. J. Physiol. 261
(Renal Fluid Electrolyte Physiol. 30): F1095-FllOl, 1991.Both mitogenic and inflammatory phenomenaaccompany the renal responseto ischemicinjury. Previous studieshave shown that several nuclear-binding membersof the immediate early genesare prominently expressedafter renal ischemiaand may underlie the mitogenic responseto such injury. We now report on the expressionof JE and KC, other growth-factor-responsive genesthat codefor small secretedglycoproteins with cytokinelike properties, which may play a role in inflammation. The expressionof the immediateearly genesJE and KC was determined in rat kidney tissue at varying time points after release of a 50-min period of bilateral renal hilar clamping. Relative levelsof mRNA for JE and KC were analyzed by Northern blot analysis of cortical and outer stripe mRNA. KC mRNA rose rapidly to peak values at 1 h and returned toward low baseline levels by 24 h after releaseof the hilar clamp. By contrast, JE mRNA reachedpeak levels later and remained elevated for at least 96 h after ischemia.JE antigen was localized immunocytochemically to the apical regionsof the cortical and medullary thick ascendinglimbs as well as in the lumen of the distal nephron in ischemic kidneys. Cells of the glomerulus and proximal tubules were negative for JE antigen. In contrast to the increasein JE and KC mRNA, steady-state levels of uromodulin (Tamm Horsfall) mRNA, a cytokine binding protein also made by the thick ascending limb, declined to virtually undetectable levels by 24 h after ischemia.Thus the increases in JE and KC are not generalized phenomena.These studies demonstratethat the kidney rapidly expressesgenesencoding leukocyte chemoattractants in responseto ischemia. JE and KC may serve the renal regenerative responseeither by promoting the movement of cells along the basementmembrane of injured tubules or by attracting leukocytes into the ischemic kidney. The decline of the cytokine binding Tamm-Horsfall protein may also increasethe activity of locally generated or filtered cytokines within the kidney. ischemic renal failure; immediate early genes JE and KC; Tamm-Horsfall; cytokines and regeneration
and nephrotoxic acute renal failure requires replacement of dead tubular cells with living ones that restore the continuity of the renal epithelium (30). During this process normally quiescent renal cells increase their nucleic acid synthesis and RECOVERY FROM ISCHEMIC
0363-6127/91
$1.50
undergo mitosis (30). These phenomena occur in most forms of acute renal injury, suggesting a similar underlying mechanism. Entry into the cell cycle is largely coordinated by changes in gene expression. Growth-promoting signals reach the nucleus by a variety of intracellular pathways and induce the transcription of many genes (29). First among them are the so-called immediate early genes, whose increased expression begins almost immediately after such stimulation, lasts only briefly, and does not depend on new protein synthesis (1). Some of these immediate early genes code for nuclear or DNA binding proteins that presumably initiate a genetic cascade ending in cell division. The transcription of these genes is increased by ischemic renal damage in a manner that is strikingly similar to that induced by growth factor stimulation of quiescent cells in culture. The prototype of the nuclear binding members of the immediate early genes, c-fos mRNA, increases almost immediately after release of renal hilar clamping (19, 26). Its induction is short lived and precedes the peak of renal DNA synthesis. A similar increase is also observed for the immediate early gene Egr 1 (19, 26). It is highly likely, therefore, that the immediate early genes somehow serve the renal regenerative response to ischemia. Renal injury also has profound effects on the production of epidermal growth factor (EGF). Ischemia and cisplatin reduce preproEGF mRNA and reduce EGF excretion (26, 27) but, in contrast to the changes in the immediate early genes, preproEGF mRNA levels fall more gradually and remain low for a prolonged period of time. The renal failure-induced reduction of preproEGF mRNA is of interest, since it cannot be ascribed to the simple loss of the cells that produce it and because the thick ascending limb and distal tubule, which are its only sites of synthesis, undergo morphological changes that are mild, reversible, and transient after each of these insults (6). The movement of regenerating cells along the basement membrane of injured tubules (30) and the accumulation of leukocytes into the kidney (17) are important responses to renal ischemia. Although their numbers are small, recent studies on the role of leukocytes during renal ischemia (13, 17) show them to be important determinants of the functional and morphological manifes-
Copyright 0 1991 the American Physiological Society
F1095
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on September 7, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
F1096
JE AND
KC
EXPRESSION
tations of ischemic renal failure. Possible mediators of these events are unknown. The genes JE and KC, originally identified as immediate early genes induced by platelet-derived growth factor in mouse 3T3 cells, appear to code for cytokines more involved in inflammation rather than growth. Sequence and expression analysis shows that unlike other early response genes, such as cfos, JE and KC encode secreted glycoproteins (18, 23). The only known function of the JE and KC products is chemotaxis. The KC protein is chemotactic for neutrophils (31) and is a member of a superfamily of proteins that includes platelet factor 4 and connective tissueactivating peptide III. JE is chemotactic for monocytes (32) and smooth muscle cells (15) and is the rat homologue of the human monocyte-specific chemotactic factor MCP-1 (32). These observations suggest that the JE and KC products are linked in some fashion to wound healing and inflammatory responses (31). Increased expression of these genes has also recently been observed after vessel wall injury (15), where it is speculated that they may play an important role in the inflammatory response to such injury in vivo. To determine whether the expression of KC and JE was increased by renal ischemia, relative levels of their mRNAs were determined by Northern analysis at varying times after a 50-min period of renal hilar clamping. Because of the cytokine characteristics of these proteins, we also examined the expression of the renal cytokine binding Tamm-Horsfall (TH) protein, whose cDNA structure and renal site of synthesis are similar to preproEGF (9). The results show that the expression of JE and KC increases and TH decreases after renal ischemia. Immunocytochemical studies show that JE product is localized to the thick ascending limb and distal tubule, the same sites of EGF and TH production and demonstrate that the thick ascending limb is a prominent site of cellular reaction to renal ischemic damage. METHODS
DURING
RENAL
ISCHEMIA
Poly(A)+ RNA was isolated by oligo(dT)-cellulose (type 3; Collaborative Research, Bedford, MA) chromatography (18). Poly(A)’ RNA (5 and 7.5 pg/lane) was electrophoresed through formaldehyde-l% agarose gels (12) and transferred overnight to nitrocellulose membranes (BA 85, Schleicher and Schuell, Keene, NH) as described (14). Hybridization was carried out with nick-translated DNAs labeled with [32P]deoxycytidine 5’-triphosphate (dCTP) (500 Ci/mmol) to a sp act of - 5 x lo8 counts/ min (cpm)/pg DNA (22). Nitrocellulose blots were prehybridized at 45°C overnight with denatured salmon sperm DNA (0.1 mg/ml) in 5 X saline sodium citrate (SSC), 50% deionized formamide, and 0.02% Denhardt’s solution [ 0.02% Ficoll, 0.02% polyvinylpyrrolidine, and 0.02% bovine serum albumin (BSA)] and hybridized at 45°C overnight in the same solution containing 10% dextran sulfate and radiolabeled probe. Nonspecifically bound radioactivity was removed by extensive washing as described (33). After washing, the blots were air dried and exposed overnight to Kodak XAR film at -70” with intensifying screens. Quantitative variabilities of isolation and transfer of RNAs were accounted for by reprobing the Northern blots with a cDNA probe for glyceraldehyde-3-phosphate-dehydrogenase. The sequences used for nick translation were excised with appropriate restriction endonucleases and purified by preparative gel electrophoresis. The cDNAs for JE [a 750-base pair (bp) Pst I fragment of pBC-JE]; KC (a 820-bp Pst I fragment of pKC); TH (a 2.2-kb Eco RI fragment in pBR322 (20); murine preproEGF (a 700-bp Pst I fragment of pmegfl0 (21)); and rat glyceraldehyde-3-phosphate-dehydrogenase from prGAPDH-13 (7) were gifts of the authors. To quantify levels of JE, KC and TH mRNA, autoradiograms were scanned in two dimensions using an Apple scanner and densitometry employing 256 gray scales on an Apple Macintosh IIcx computer using Image 1.36 software (Wayne Rasband, National Institutes of Health) and related to the strength of the GAPDH signal on the same blot.
General Methods
Immunocytochemical localization of JE during acute renal failure. Kidneys were removed at varying times
Animal preparation. Male Sprague-Dawley rats weighing 250-500 g were fed a standard rat chow (Rat Chow 5012; Ralston Purina, St. Louis MO, containing 22.8% protein, 4.5% fat, and 4.6% fiber and water) and offered tap water ad libitum. After a period of 4-6 days on this diet, rats were anesthetized (ketamine, 75 mg/kg), and both renal arteries were occluded for 50 min under sterile conditions. In sham-operated animals, anesthesia, manipulation of the renal hila, and exposure of the peritoneal cavity for 50 min were performed as in the ischemia studies, but occlusion of the renal arteries was omitted. Animals were killed at various times in the reflow period as required by the specific protocols. RNA blot analysis. Relative levels of mRNA were analyzed by Northern and dot-blot analysis. Total RNA was isolated from the cortex and outer stripe of the outer medulla from anesthetized animals at varying time periods after renal ischemia or sham operation by the guanidinium thiocyanate procedure (5) after intra-arterial perfusion of the kidney with cold 154 mM NaCl-50 mM tris(hydroxymethyl)aminomethane (Tris) (pH 7.4).
after release of the renal hilar clamp and after intravascular perfusion with freshly prepared 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Small sagittal sections (l-2 mm in thickness) were cut and postperfusion fixed for no longer than 30 min in the same buffered paraformaldehyde solution. After several washes with phosphate-buffered saline (PBS) (NaC1154 mM, sodium phosphate, 100 mM, pH 7.3) and a 30-min period of cryoprotection in 20% sucrose, the tissue was snap frozen in liquid nitrogen. Thin (6-8 pm) sections were cut in a microtome and placed onto slides previously treated with poly-L-lysine to promote better section adherence. Sections were stored at -70°C before proceeding to the immunocytochemical steps. On removal from storage, the sections were fixed an additional 10 min with 4% paraformaldehyde in 0.5 M Tris (pH 7.6) and rinsed 3 times in 0.5 M Tris buffer. The sections were then treated with 0.3% hydrogen peroxide in methanol for 20 min to reduce nonspecific background staining by endogenous peroxidase activity and rinsed three times in 0.5 M Tris. To block nonspe-
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on September 7, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
I_
.
.
*-
JJ!S ANLJ
*.,-.
-vv.-.---AI-.
KU
.
DURING
RENAL
F1097
ISCHEMIA
k!Xk'lt.l~LZS5lUN
TABLE
1. Effect of ischemia on renal KC and JE mRNA
No. of h after Control 1 4 24 48 98 168
KC
n
JE
n
23+13 127f31* 75*13t 45k12 ND 11*15 ND
4 4 4 4
19+s 43*15 56+6$ 73-t23t Sl+S§ 69+6§ 41+14
6 3 6 6 3 3 2
ischemia
2
Values are means f SE; n = no. of determinations. Densitometric readings for KC and JE mRNA were divided by GAPDH readings and are in percent. ND, not determined. *P < 0.025; t P < 0.05, $ P < 0.005, and $ P < 0.001, significantly different from control.
FIG. 1. Northern analysis of KC mRNA. Poly(A)+ RNA (5 pg/lane) isolated from male Sprague-Dawley rat kidney cortex was applied to each lane from control (1st lane) and ischemic animals (Zones 2-4) at times indicated. Location of 18s RNA is shown, Z& here and in Figs. 2-5. KC (top) and GAPDH (bottom) cDNA probes were used as described in METHODS.
TH GAPDH FIG. 4. Northern analysis of TH mRNA. Poly(A)+ RNA (5 pg/lane) was applied to each lane from control (1st lane) and ischemic animals (lanes 2-6) at times indicated. TH and GAPDH cDNA probes were used as described in METHODS.
FIG. 2. Northern analysis of JE mRNA. Poly(A)+ RNA (7.5 rg/ lane) was applied to each lane from control (1st lane) and ischemic animals (.&es 2-6) at times indicated. JE and GAPDH cDNA probes were used as described in METHODS.
--*-- KC !
-
JE
:
0
24
48
Time
72
96
120
144
166
(hrs)
FIG. 3. Relative levels of JE and KC mRNA in ischemic kidneys. JE and KC mRNA levels were determined by 2-dimensional densitometry of scanned audioradiograms. Levels were normalized to those of GAPDH mRNA and are expressed relative to control nonischemic kidney levels where control = 1. Each point represents mean of at least 3 animals.
with a specific anti-JE serum at 1:200 dilution in TGBA containing the same concentration of goat serum and biotin overnight at 4°C. The anti-JE serum was raised in New Zealand White rabbits against mouse JE by methods previously described (24). The slides were then rinsed in 0.5 M Tris and incubated with biotinolated anti-rabbit immunoglobulin G (IgG) (Amersham) for 1 h at room temperature. The slides were then rinsed in 0.5 M Tris three times and incubated further in peroxidase-conjugated streptavidin (Jackson Immunoresearch Labs, West Grove, PA) diluted 1:lOO for 1 h at room temperature. After slides were rinsed in 0.5 M Tris the reaction was developed in 0.05% 3,3’diaminobenzidine and 0.01% hydrogen peroxide in 0.5 M Tris for 5 min and followed by a brief wash in 0.5 M Tris and water and dehydrated in graded alcohols and xylene. After counterstaining with hematoxylin, the slides were mounted with a cover slip and observed under a light microscope. The intensity of the immunohistochemical staining was scored using the following criteria: 0 = no detectable localization; + = weak, ++ = moderate and +++ = strong staining. Sections from control animals, as well as substitution of the primary antisera with nonimmune serum, were used as negative controls. RESULTS
Figure 1 is a representative Northern blot anlysis of from normal and ischemic animals. KCspecific mRNA was present at very low levels in normal kidneys. Levels of KC mRNA were markedly induced by renal &hernia, peaking between 1 and 4 h. Levels returned to baseline within 24 h and remained low for up to 1 wk after ischemia. This rapid induction and fall of KC mRNA
cific antibody binding sites the sections were incubated with nonimmune goat serum (20%) and biotin (0.1%) in Tris-gelatin-bovine albumin (TGBA) buffer (gelatin 0.1%; BSA 0.1%; NaN3 0.1% in 0.5 M Tris (pH 7.6) for 1 h at room temperature. Then the section was incubated
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on September 7, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on September 7, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
JE
AND
KC
EXPRESSION
KC mRNA conforms to the general characteristics of immediate early gene expression (1). Figure 2 shows results of Northern blot analysis of JE mRNA. JE mRNA was also present at low levels in control kidneys. Like KC, JE mRNA was rapidly induced following renal ischemia. However, unlike KC, elevated levels of edE mRNA persisted for up to 1 wk, peaking at between 48-96 h. Figure 3 is graphic representation of densitometric analysis of KC and JE mRNA levels pooled from multiple experiments (Table 1). Levels of JE and KC were normalized to GAPDH and are expressed as arbitrary units relative to a control of one for nonischemic kidneys. Each time point is an average of at least three individual animals. KC mRNA peaked at 4.8 t 1.8fold above control at 1 h (n = 3, P ~0.025). JE mRNA increased to 2.9 & O&fold above control (n = 6, P CO.005) at 4 h and peaked at 4.3 & 0.6 fold (n = 3, P CO.001) above control levels at 48 h after release of the hilar clamp. Figure 4 shows representative Northern blot analysis of 2X mRNA. In contrast to JE and KC mRNA, TH mRNA is high in normal kidney. Beginning at -6 h (data not shown), TH mRNA levels fell progressively to only 21% of control at 24 h and 10% of control at 48 h. TH mRNA levels were still greatly reduced at 96 h (21% of control values) but returned to control levels 1 wk after ischemia. In cell culture, JE protein is rapidly secreted after its mRNA is induced (23, and unpublished observations). In addition, JE protein is present in a variety of cells including fibroblasts (24)) vascular smooth muscle (15)) and endothelial cells (25). Thus it was of interest to determine the precise localization of the JE product in the kidney. Using an anti-JE antiserum that identifies a single protein on Western blots (24), we performed immunocytochemical studies of renal tissue at early time points following the induction of JE. The results of these immunocytochemical studies of JE protein localization are shown in Fig. 5. Kidney sections of the outer stripe of the outer medulla in sham-operated control (A and C) and in 4-h postischemic animals (B and D) are shown. In both unstained and stained sections of the kidney (B and D) JE protein, indicated by the arrows, was seen at the luminal aspect of the thick ascending limbs of Henle’s loop in the ischemic kidneys and was not detectable in the control kidneys (A and C). No reaction product was detected in glomeruli or proximal tubules. Treatment of slides with 0.3% hydrogen peroxide to reduce endogenous peroxidase activity and nonimmune goat serum to block nonspecific antibody binding sites was used to reduce background activity. Incubation with nonimmune rabbit serum did not reveal specific staining. DISCUSSION
The present studies show that during the early reflow period of renal ischemia, the expression of JE and KC,
DURING
RENAL
ISCHEMIA
F1099
two immediate early genes induced by growth factor and lipopolysaccharide addition to cells in culture, is increased. But unlike other immediate early genes such as c-fos and Egr-1, whose expression is also increased following renal ischemia, the increased expression of JE was prolonged and peaked at least 4 days after ischemia. Such prolonged expression of JE by the ischemia-damaged kidney is unique. Mitogenic (18, 23) and lipopolysaccharide-stimulated (10) expression of JE in cell culture is short lived. In addition, we have recently reported that balloon injury of rat and rabbit aorta (15) induced a transient rise in JE mRNA levels. This suggests that the mechanism for induction and regulation of JE mRNA in the kidney may be different from that in balloon-injured vessels and distinct from those previously examined in cell culture. In addition the elevated levels of JE mRNA persisted well beyond the ischemiainduced peak of DNA synthesis that occurs at 24 h after induction of ischemia (26). These observations coupled with the localization of JE to the thick ascending limb, a site not prominent for cell division after ischemia (6), would indicate that JE and probably KC serve other roles in renal ischemia not specifically linked to cell division. What these roles are can only be speculated on at the present time, but the chemotactic and inflammatory properties of JE and KC would suggest that they may promote the movement of regenerating cells to restore the integrity of the tubule epithelium or promote the accumulation of leukocytes into the ischemic kidney (17). The coincidental fall in TH expression after ischemia would also enhance the activity of locally produced or filtered cytokines, if, as suggested, it functions as a cytokine-binding and inactivating protein (20). Thus the reciprocal changes in JE, KC, and TH may modulate the inflammatory response to renal ischemia. Such enhancement in cytokine activity would also be expected to have effects on renal function (28). JE antigen was immunocytochemically localized to the thick ascending limb and distal tubule of ischemic kidneys. It is important to note that finding the JE protein on the luminal surface of the thick ascending limb does not necessarily indicate that these cells produce it. JE may be made elsewhere and bind to sites on these cells. However, in the absence of significant JE antigen elsewhere in the kidney, the thick ascending limb should be considered the most likely site of JE production. Previous studies have established that the thick ascending limb undergoes prominent changes in gene expression during renal ischemia, both positive and negative. Epidermal growth factor mRNA, made only by the thick ascending limb and distal tubule of normal kidneys (24) falls markedly during renal ischemia (26), whereas the product of Egr-1, a nuclear binding protein that increases after renal ischemia, is also found in the nuclei of the thick ascending limb (3). The present studies demonstrate that ischemia induces a fall in levels of TH mRNA, also produced by the thick ascending limb (5). The pathophysiological
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on September 7, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
FllOO
JE
AND
KC
EXPRESSION
DURING
significance of these changes in thick ascending limb transcriptional activity is unknown, but recent studies on the isolated perfused kidney have focused attention on the possible role of the thick ascending limb in the pathogenesis of acute renal failure (4). The consequences of the altered expression of genes by the thick ascending limb remains to be established, but defects in salt transport have been documented in apparently morphologically intact thick ascending limbs studied in situ (16) or by perfusion of isolated segments of thick ascending limbs of ischemic animals (8). The thick ascending limb may be a crucial element in the renal response to ischemia as these rapid and long lasting changes in gene expression would indicate. Studies designed to identify how these genes are regulated and how to alter their expression should provide important clues to their functional significance and lead to novel strategies designed to maximize recovery from renal failure. We thank Dr. George Acs for his helpful suggestions during the course of these studies. This work was supported in part by a grant from the National Kidney Foundation of New York-New Jersey to S. Saggi, National Institutes of Health Grants HL-43302 and CA-53901, and a grant-inaid from the American Heart Association, New York Affiliate to M. Taubman. This is publication No. 48 from the Brookdale Center for Molecular Biology. Address for reprint requests: R. Safirstein, Dept. of Medicine, Renal Division, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029 Received 12 December 1990; accepted in final form 24 July 1991. REFERENCES H. MCDONALD-BRAVO, J. Complexity of the early genetic response to growth factors in mouse fibroblasts. 1Mol. Cell. Biol. 8: 2140-2148, 1988. 2. AVIV, H., AND P. LEDER. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc. Nutl. Acud. Sci. USA 69: 1408-1412, 1972. 1. ALMENDRAL, BURCKHARDT,
J. M., D. SOMMER, J.PERERA,
AND R. BRAVO.
3. BONVENTRE, J. V., A. J. OUELLETTE, BROWN. Localization of the EGR-1
V. P. SUKHATME,
AND D.
protein product to the distal nephron after renal ischemia and reperfusion (Abstract). CZin. Res.
38: 442A, 1990. 4. BREZIS, M., S. ROSEN,
F. H. EPSTEIN. Selective vulnerability of the medullary thick ascending limb to anoxia in the isolated perfused rat kidney. J. CZin. Inuest. 73: 182-190, 1983. 5. CHIRGWIN, J. M., A. E. PRZYBYLA, R. J. MACDONALD, AND W. J. RUTTER. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294-5299,1979. P. SILVA,
AND
6. DONOHOE, J. F., M. A. VENKATACHALAM, N. G. LEVINSKY. Tubular leakage and
ischemia: structural-functional
D. B. BERNARD,
AND
obstruction after renal correlations. Kidney Int. 13: 208-
222,1978. 7. FORT, P., L. MARTY, P. JEANTEUR, AND
M. PIECHACZYK, S. E. SABROUTY, C. DANI, J. M. BLANCHARD. Various rat adult tissue express only one major mRNA species from glyceraldehyde-3phosphate-dehydrogenase multigenic family. NucZ. Acid Res. 13:
1431-1442,1985. 8. HANLEY, M.
J. Isolated nephron segments in a rabbit model of ischemic acute renal failure. Am. J. Physiol. 239 (Renal Fluid Electrolyte Physiol. 8): F17-F23, 1980. 9. HESSION, C., J. M. DECKER, A. P. SHERBLOM, S. KUMAR, C. C. YUE, R. J. MATTALIANO, R. TIGARD. E. KAWASHIMA, U. SCHMEISSNER, S. HELETKY ET AL. Uromodulin (Tamm-Horsfall glycoprotein): A renal ligand for lymphokines. Science Wash. DC 237: 1479-1484,1987. 10. INTRONA, M., R. C. BART, ILTON, AND D. 0. ADAMS.
JR., C. S. TANNENBAUM, T. A. HAMThe effect of LPS on exnression of the
RENAL
ISCHEMIA
early “competence” genes JE and KC in murine peritoneal macrophages. J. Immunol. 138: 3891-3896, 1987. 11. KLAUSNER, J. M., I. S. PATERSON, G. GOLDMAN, L. KOBZIK, C. RODZEN, R. LAWRENCE, C. R. VALERI, D. SHEPRO, AND H. B. HECHTMAN. Postischemic renal injury is mediated by neutrophils and leukotrienes. Am. J. Physiol. 256 (Renal Fluid Electrolyte Physiol. 25): F794-F802, 1989. 12. LEHRACH, H., D. DIAMOND, J.
M. WOZNEY, AND H. BOEDTKER. RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry 16:
4743-4751,1977. 13. LINAS, S. L., J. E. REPINE.
P. F. SHANLEY, D. WHITTENBURG, E. BERGER, AND Neutrophils accentuate ischemia reperfusion injury in isolated rat kidneys. Am. J. Physiol. 255 (Renal Fluid Electrolyte
Physiol. 24): F728-F735, 1985. 14. MANIATIS, G. E., F. FRITSCH, AND J. SAMBROOK. Molecular CZoning Manual. New York: Cold Spring Harbor, 1982, p. 383-386. 15. MARMUR, J. D., V. 1;. FRIEDRICH, JR., M. ROSSIKHINA, B. J. ROLLINS, AND M. B. TAUBMAN. The JE gene encodes smooth
muscle chemotactic factor that is induced by vascular injury (Abstract). Circulation 82: 698, 1990. 16. MASON, J., H.-U. GUTSCHE, L. MOORE, AND R. MULLER-SUUR. The early phase of experimental acute renal failure IV. The diluting ability of the short loops of Henle. Pfluegers Arch. 279: 11-18, 1979. 17. OLOF, P., A. HELLBERG, 0. T. KALLSKOG, G. OJTEG, AND M. WOLGAST. Peritubular capillary permeability and intravascular RBC aggregation after ischemia: effects of neutrophils. Am. J. Physiol. 258 (Renal Fluid Electrolyte Physiol. 27): FlO18-F1025, 1990. 18. OQUENDO, P., J. ALBERTA, W. DUANZHI, J. C. GRAYCAR, R. DERYNCK, AND C. D. STILES. The platelet- derived growth factorinducible KC gene encodes a secretory protein related to platelet a-granule proteins. J. BioZ. Chem. 264: 4133-4137, 1989. 19. OUELLETTE, BONVENTRE.
A. J., R. A. MALT,
V. P. SUKHATME,
AND J. V.
Expression of two “immediate early genes, Egr-1 and c-fos, in response to renal ischemia and during renal hypertrophy in mice. J. CZin. Invest. 85: 766-771, 1990. PENNICA, D., W. J. KOHR, W.-J., KUANG, D. GLAISTER, B. B. AGGARWAL, E. CHEN, AND D. V. GOEDDEL. Identification of human uromodulin as the Tamm-Horsfall urinary glycoprotein.
21 Science Wash. DC 236: 83-88, 1987. . RALL, L. B., J. SCOTT, B. I. BELL, R. J. CRAWFORD, J. D. PENSCHOW, H. D. NIALL, AND J. P. COGHLAN. Mouse prepro-epiderma1 growth factor synthesis by the kidney and other tissues. Nature Lord. 313: 228-231,1985. 22* RIGBY, P. W. J., M. DIECKMAN,
C. RHODES, AND P. I. BERG. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. BioZ. 113: 237251,1977.
AND C. D. STILES. Cloning and 23* ROLLINS, B. J., E. D. MORRISON, expression of JE, a gene inducible by platelet-derived growth factor and whose product has cytokine-like properties. Biochemistry 85: 3738-3742,1988.
24* ROLLINS, B. J., P. STIER, T. ERNST, AND G. G. WONG. The human homolog of the JE gene encodes a monocyte secretory protein. MOL. CeZZ. BioZ. 9: 4687-4695,
1989.
E. J. LEONARD, AND J. S. POBER. 25. ROLLINS B. J., T. YOSHIMURA, Cytokine-activated human endothelial cells synthesize and secrete a monocyte chemotractant, MCP-l/JE. Am. J. Path. 136: 12291233,199O.
R., P. M. PRICE, S. J. SAGGI, AND R. C. HARRIS. 26. SAFIRSTEIN, Changes in gene expression after temporary renal ischemia. Kidney Int. 37: 1515-1521,199O. 27. SAFIRSTEIN, R., A. ZELENT,
mRNA and diminished Kidney Int. 28. SCHREINER,
AND P. M. PRICE. Reduced preproEGF EGF excretion during acute renal failure.
36: 810-815,1989. G. F., AND D. E. KOHAN.
Regulation of renal transport and hemodynamics by macrophages and lymphocytes. Am. J. Physiol. 258 (Renal
FZuid
Electrolyte
Physiol.
27): F761-F767,
1990.
29. STILES C. D. The biologic role of oncogenes- insights from plateletderived growth factor. Cancer Res. 45: 5215-5218,1985. 30. TOBACK, F. G. Control of renal regeneration after acute tubule necrosis. In: NephroZogy, edited by R. R. Robinson. New York: &ringer-Verlae. 1984. D. 748-762.
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on September 7, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
JE AND KC EXPRESSION 31. WATANABE,
K., K. KONISHI, M. FUJIOKA, S. KINOSHITA, AND H. The neutrophil chemoattractant produced by the rat kidney epithelioid cell line NRK-52E is a protein related to the KC/gro protein. J. Biol. Chem. 264: 19559-19563, 1989. 32. YOSHIMURA, T., AND E. J. LEONARD. Secretion by human fibroblasts of monocyte chemoattractant protein-l, the product of gene NAKAGAWA.
DURING
RENAL
ISCHEMIA
FllOl
JE. J. Immunol. 144: 2377-2383,199O. 33. ZELENT, A. Z., M. A. SELLS, P. M. PRICE, A. MOHAMAD, G. Acs, AND J. K. CHRISTMAN. Murine cells carrying integrated genomes of hepatitis B virus DNA transcribe RNA from endogenous promoters on both viral strands and express middle and major viral envelope proteins. J. Viral. 61: 1108-1115, 1987.
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on September 7, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
