Leukemia Inhibitory Factor Is Involved in Tubular Regeneration after Experimental Acute Renal Failure
Jun Yoshino,
Toshiaki Monkawa,
Mihoko Tsuji,
Matsuhiko Hayashi and
Takao Saruta
Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan.
Correspondence to Dr. Toshiaki Monkawa, Department of Internal Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160–8582, Japan. Phone: 81-3-5363-3796; Fax: 81-3-3359-2745; E-mail: monkawa{at}sc.itc.keio.ac.jp
ABSTRACT. Leukemia inhibitory factor (LIF) is known to playa crucial role in the conversion of mesenchyme into epitheliumduring nephrogenesis. This study was carried out to test thehypothesis that LIF and LIF receptor (LIFR) are involved inthe renal epithelial regeneration after acute renal failure.First, the authors investigated the spatiotemporal expressionof LIF and LIFR in fetal and adult rat kidney. In developingkidney, LIF was expressed in the ureteric buds and LIFR waslocated in nephrogenic mesenchyme and the ureteric buds; inadult kidney, LIF and LIFR expression was confined to the collectingducts. Next, the authors examined the expression of LIF andLIFR during the recovery phase after ischemia-reperfusion injury.Real-time PCR analysis revealed that LIF mRNA expression wassignificantly increased from day 1 to day 7 after reperfusionand that LIFR mRNA was upregulated from day 4 to day 14. Histologicanalysis demonstrated that the increased expression of LIF mRNAand protein was most marked in the outer medulla, especiallyin the S3 segment of the proximal tubules. To elucidate themitogenic role of LIF in the regeneration process, culturedrat renal epithelial (NRK 52E) cells were subjected to ATP depletion(an in vitro model of acute renal failure), and LIF expressionwas found to be enhanced during recovery after ATP depletion.Blockade of endogenous LIF with a neutralizing antibody significantlyreduced the cell number and DNA synthesis during the recoveryperiod. These results suggest that LIF participates in the regenerationprocess after tubular injury.
The mammalian kidney is susceptible to injury by ischemia andnephrotoxins, and recovery of normal renal function requiresregeneration of damaged tubular epithelium. The process of regenerationafter renal injury is characterized by a sequence of eventsthat includes epithelial cell spreading, migration to coverexposed areas of the basement membrane, cell dedifferentiationand proliferation to restore cell number, and then differentiation(1,2). In many respects this nephrogenic repair process resemblesthe growth and maturation of nephrons during kidney development(3,4). Several genes critical for kidney development have beenshown to be upregulated in the regeneration process after injuryand participate in the regeneration. Pax-2 (5), Wnt-4 (6), andactivin-A (7) have been shown to be re-expressed in the regeneratingtubules after injury (8–11). These findings support thehypothesis that a cascade of developmental gene pathways isreactivated during tissue regeneration.
Leukemia inhibitory factor (LIF), a member of the interleukin-6(IL-6) family, is a multifunctional cytokine originally identifiedas a proliferation inhibitor and differentiation inducer ofmouse myeloid leukemia cell line M1 (12,13). LIF is synthesizedby a variety of cells, including renal epithelial cells (14),and, functionally, it has been implicated in a number of processesincluding development, hematopoiesis, inflammation, and regenerationafter injury (15). LIF has been shown to play a pivotal rolein kidney development. It is secreted by ureteric buds and inducesconversion of mesenchyme into epithelium (16).
The importance of LIF in nephrogenesis prompted us to test thehypothesis that LIF participates in renal epithelial tubularregeneration. In this study, we first investigated the expressionof LIF and LIF receptor (LIFR) in ischemia/reperfusion–injuredkidney and in normal fetal and adult rat kidney; we then usedan in vitro ischemia model (ATP depletion method) to investigatethe mitogenic effect of LIF on kidney epithelial cells (NRK52E) during the regenerative process. The results showed thatLIF is transiently increased in regenerating tubular cells duringthe recovery phase both in vivo and in vitro and that LIF contributesto renal epithelial regeneration.
Animals
Sprague-Dawley rats were obtained from Charles River Japan (Tokyo,Japan) and anesthetized by intraperitoneal injection of pentobarbitalbefore use in the experiments. All procedures employed in theanimal experiments were conducted in accordance with the Guidelinefor the Care and Use of Laboratory Animals of Keio UniversitySchool of Medicine.
Bilateral ischemic renal injury was produced in male rats weighing200 to 230 g by clamping both renal arteries for 45 min. Theclamp was then removed to allow reperfusion, and rats were killedafter 1, 2, 4, 7, 14, and 30 d of reperfusion (n = 5 or 6 pergroup).
For histologic assessment, fetal and adult kidneys were fixedin 10% phosphate-buffered formalin, embedded in paraffin, andcut serially into 5-µm-thick sections.
Real-Time PCR
Total RNA was isolated from kidney homogenates and culturedcells with the TRIzol Reagent (Invitrogen, Carlsbad, CA). First-strandcDNA was synthesized from total RNA by using random hexamers.Real-time PCR was performed on a TaqMan ABI 7700 Sequence DetectionSystem (Applied Biosystems, Foster City, CA). PCR amplificationwas performed using TaqMan PCR Universal Master Mix (AppliedBiosystems). Thermal cycler conditions consisted of holdingat 50°C for 2 min and 95°C for 10 min, followed by 30–40cycles of 95°C for 15 s and 60°C for 1 min. The followingoligonucleotide primers (50 nM) and probes (200 nM) were used:rat LIF (GenBank accession number AB010275): sense, 5'-CAGTGCCAATGCCCTCTTTA-3',antisense, 5'-GCATGGAAAGGTGGGAAATC-3'; internal fluorescence-labeledTaqMan MGB probe (FAM): 5'-CCCAACAACGTGGATAAGCTATGTGCGC-3';rat LIF receptor (GenBank accession number D86345): sense, 5'-TCATCAGTGTGGTGGCAAGAA-3', antisense, 5'- TCATCAGTGTGGTGGCAAGAA-3';internal fluorescence-labeled TaqMan probe (FAM): 5'-TTCTGCCGGTTCATCTCCACCTTCAA-3'.Gene expression of the target sequence was normalized in relationto expression of 18S ribosomal RNA as an endogenous control.Each sample was tested in duplicate. Results are expressed relativeto data from pre-ischemic kidneys that were arbitrarily assigneda value of 1.
In Situ Hybridization
A cDNA for rat LIF was generated by reverse transcription-PCRwith the following primers: sense, 5'-TGCCCCTACTGCTCATTCTG-3',antisense, 5'-GACACAGGGCACATCCACAT-3'. The amplified PCR fragmentwas initially ligated to pCR II vector (Invitrogen) and digoxigenin(DIG)–labeled antisense, and sense cRNA probes were synthesizedwith T7 or SP6 RNA polymerases (DIG RNA labeling kit SP6/T7;Roche Diagnostics, Indianapolis, IN). Tissue sections were deparaffinized,permeablized with proteinase K, and refixed with 4% paraformaldehydein phosphate-buffered saline (PBS). The prehybridization andhybridization steps were carried out at 65°C for 2 h and12 h, respectively. The hybridization buffer was composed of50% formamide, 5 x SSC, 5 x Denhardt’s solution, 250 µg/mlBaker’s yeast tRNA, 500 µg/ml salmon sperm DNA,and cRNA probes. After post-hybridization washing, sectionswere incubated with anti-digoxigenin antiserum conjugated toalkaline phosphatase, and histochemical detection was performedusing a 4-nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphatemixture (Roche Diagnostics). The same procedure performed withthe sense cRNA probe served as a negative control.
Immunohistochemical Staining
After deparaffinization, sections were treated with 0.3% hydrogenperoxidase in methanol for 30 min to remove endogenous peroxidaseactivity. They were then blocked in normal horse serum and incubatedat 4°C overnight with the primary antibodies: anti-mouseLIF antibody (R&D Systems, Minneapolis, MN), anti-humanLIFR antibody (Santa Cruz Biotechnology, Santa Cruz, CA), andanti-human aquaporin-1 (AQP-1) antibody (Alomone Labs, Jerusalem,Israel). Next, the sections were incubated with biotinylatedsecondary antibody and incubated with avidin-biotin horseradishperoxidase complex (Vectastain Elite ABC kit; Vector Laboratories,Burlingame, CA). The sections were visualized with a VectorDAB peroxidase substrate kit (Vector Laboratories) and werecounterstained with hematoxylin. As a negative control, theprimary antibody was replaced with normal horse serum, and nopositive immunostaining was observed.
For 5-bromo-2'-deoxyuridine (BrdU) staining, rats were intraperitoneallyinjected with BrdU 2 h before sacrifice. Formalin-fixed paraffinsections were subjected to BrdU staining by using a Cell ProliferationKit (Amersham Biosciences, Piscataway, NJ) according to themanufacturer’s instructions. In brief, after deparaffinization,sections were treated with 0.3% hydrogen peroxidase in methanolfor 30 min to remove endogenous peroxidase activity. Tissuesections were then placed in citrate buffer and heated for 10min in a microwave oven; after incubating them with anti-BrdUantibody, the sections were incubated with HRP-conjugated IgGand developed with a Vector DAB peroxidase substrate kit.
Double immunohistochemistry was performed to detect LIF expressionin combination with the proximal tubular marker AQP-1 (17,18).The first immunohistochemical staining was performed with aVector VIP substrate kit (Vector Laboratories) to obtain a purplereaction product, and the sections were then washed and blockedwith an avidin-biotin blocking kit (Vector Laboratories). Thesecond immunostaining was performed with a Vector SG substratekit (Vector Laboratories) to obtain a blue reaction product.
Cell Culture
A normal rat kidney epithelial-derived cell line, NRK 52E (19),was grown in Dulbecco modified essential medium with 10% fetalbovine serum, 100 U/ml penicillin, and 100 U/ml streptomycin,in a humidified 5% CO2/95% air environment at 37°C.
ATP Depletion Protocol
ATP depletion of NRK 52E cells was achieved by means of previouslydescribed protocols with modifications (20,21). A confluentmonolayer of NRK 52E cells was incubated in PBS with 1.5 mMCaCl2, 2 mM MgCl2, and 1 µM antimycin A (Sigma, St. Louis,MO) for 60 min. During the recovery phase after the 1-h injuryperiod, cells were repleted with ATP by incubation with a regulargrowth medium. Cellular ATP levels were determined with a luciferase-basedassay kit (Sigma).
Effect of LIF Antibody on Cell Proliferation
NRK 52E cells were subcultured in 24-well plates for cell counting,or in 96-well plates for measurement of BrdU incorporation,and subjected to ATP depletion for 1 h as described above. Afterthe injury, cells were repleted with ATP by incubation withrecovery medium containing affinity-purified anti-human LIFantibody (Genzyme, Cambridge, MA) at the concentration indicated,or with normal goat IgG as a negative control. The number ofliving cells was counted with a Cell Counting Kit-8 (Wako PureChemicals, Osaka, Japan) after the 48-h recovery phase (n =4). DNA synthesis was assessed by an enzyme-linked immunosorbentassay for BrdU (Roche Diagnostics) after the 24-h recovery phase(n = 6).
Statistical Analyses
Statistical significance was evaluated by one-way ANOVA witha Fisher’s post-hoc test. Statistical significance wasdefined as P < 0.05.
Localization of LIF and Its Receptor in Developing and Adult Rat Kidneys
The spatiotemporal expression of LIF and LIFR during kidneydevelopment was investigated by an immunohistochemical technique.At embryonic day 15 (E15), LIF expression level was very low(Figure 1A). Weak LIF immunoreactivity was observed in the uretericbuds, whereas LIFR immunoreactivity was observed in the mesenchymalcells surrounding the ureteric buds (Figure 1B). At E17, LIFimmunostaining was noted in derivatives of the ureteric buds,whereas no immunostaining was detected in the mesenchymal cells(Figure 1C). In the nephrogenic outer cortex, LIF was expressedin the tips of the branches of the ureteric buds (Figure 1D);in the medulla, intense staining was found in the major branchesof the ureteric buds and collecting ducts (Figure 1E). At E17,LIFR was expressed ubiquitously in mesenchyme, and the mostintense signal was observed in the condensing mesenchymal cellssurrounding the tips of the ureteric buds in the nephrogeniczone (Figure 1F). In addition to the mesenchymal cells, thederivatives of the ureteric buds, including ureteric bud branchtips and collecting ducts, expressed LIFR, a pattern that resembledthe distribution of LIF protein observed. The ureteric budsdifferentiated into collecting ducts, as nephrogenesis progressed.In neonatal kidneys, LIF was expressed predominantly in matureand immature collecting ducts (Figure 1G). LIFR was also expressedin mature and immature collecting ducts, whereas LIFR immunoreactivitywas still evident in mesenchymal cells in the nephrogenic cortex(Figure 1H).
Figure 1. Expression of leukemia inhibitory factor (LIF) (A, C, D, E and G) and LIF receptor (LIFR) (B, F, and H) during rat kidney development. (A and B) an E15 metanephros; (C to F) E17 fetal kidneys; (G and H) newborn kidneys (P1). (A) Faint LIF immunoreactivity was detected in the ureteric buds. (B) LIFR immunoreactivity was observed predominantly in mesenchymal cells on E15. (C) On E17, LIF was expressed in the derivatives of the ureteric buds, including the tip of ureteric buds (D), and the collecting ducts in medulla (E). (F) LIFR was expressed in mesenchymal cells and ureteric buds on E17. (G) LIF immunoreactivity was localized to the collecting ducts in newborn kidney. (H) LIFR was expressed in collecting ducts and nephrogenic mesenchyme in newborn kidney. Magnifications: x200 in A, B, C, F, G, and H; x400 in D and E.
We also localized LIF and LIFR protein in adult kidneys. Cytoplasmicimmunostaining for LIF was observed mainly in the collectingducts (Figure 2, A to C), and, as shown in Figure 2B, no LIFimmunoreactivity was detected in the S3 segment of the proximaltubules in the outer medulla. In mature kidneys, LIFR immunoreactivitywas also predominantly detected in the collecting ducts, coincidingwith the distribution of LIF protein (Figure 2, E to G). Noimmunostaining was observed in sections incubated with normalhorse serum (Figure 2, D and H).
Figure 2. Immunohistochemical localization of LIF (A to C) and LIFR (E to G) in normal adult rat kidneys. Paraffin sections from the cortex (A, D, E and H), outer medulla (B and F), and papilla (C and G) of normal adult kidneys are presented. Immunostaining of both proteins were mainly detected in the collecting ducts in the adult rat kidney. When using normal horse serum instead of the anti-LIF antibody (D) or normal horse serum instead of the anti-LIFR antibody (H), no positive signal was observed. Magnification: x200.
Expression of LIF and LIFR in the Rat Kidney after Ischemia/Reperfusion Injury
To investigate whether LIF is involved in renal tubular regeneration,we first used the real-time PCR method to quantify changes inthe expression of LIF and LIFR mRNA after ischemia/reperfusion(I/R) injury.
In post-ischemic kidneys, LIF mRNA expression had increased14.3-fold on day 1 after reperfusion, compared with its expressionin pre-ischemic kidneys (Figure 3A). The increase in LIF mRNApersisted until day 7, after which gradually returned to thebasal level by day 14. Interestingly, the increase in LIFR mRNAoccurred later than the increase in LIF mRNA (Figure 3B). TheLIFR mRNA level began to increase on day 4 and persisted untilday 14, before returning to baseline by day 30.
Figure 3. Expression of mRNA for LIF (A) and LIFR (B) in rat kidneys after ischemia/reperfusion (I/R) injury. Total RNA was extracted from kidneys at the indicated times after reperfusion. Quantification of mRNA was determined by real-time PCR. Expression was normalized to an endogenous control 18S ribosomal RNA (rRNA) and is shown as ratio to value of day 0. (A) LIF mRNA expression after I/R injury. (B) LIFR mRNA expression after I/R injury. Values represent means ± SEM for five or six animals. * P < 0.05 versus day 0.
The LIF mRNA expression in the kidneys was localized by meansof in situ hybridization. In normal kidneys, LIF mRNA appearedto be mainly present in the collecting ducts (data not shown).At 1 d after the renal I/R injury, the hybridization signalfor LIF mRNA was progressively increased, consistent with theresults of the real-time PCR analysis (Figure 4, A and B). Themost prominent increase in signal for LIF mRNA was noted inthe tubular epithelial cells in the outer medulla, which isthe site most vulnerable to ischemic injury. No positive signalwas obtained with the sense cRNA probe (Figure 4C).
Figure 4. In situ hybridization analysis for LIF mRNA in ischemic kidney. Digoxigenin-labeled antisense (A and B) or sense (C) riboprobe was used. (A and B) Strong signal for LIF mRNA was observed in tubular cells in the outer medulla of kidney 24 h after reperfusion. (C) The sense probe for LIF yielded no positive signal. Magnifications: x100 in A; x400 in B and C.
The LIF and LIFR proteins in the I/R-injured kidneys were preciselylocalized by an immunohistochemical technique. Whereas LIF proteinwas expressed in the collecting ducts in normal kidneys, increasinglyprominent expression of LIF protein was found in the outer medullabeginning 24 h after the ischemic injury (Figure 5A). Immunostainingwas particularly evident in the S3 segment of the proximal tubules,where no LIF immunoreactivity was detected under normal conditions.At higher magnification, immunoreactivity was seen in both thedetached and the attached cells in the injured tubules (Figure 5B).Double-staining was performed to detect LIF expressionin combination with aquaporin-1 (AQP-1), which is known to beexpressed in proximal tubules, especially in the S3 segment(17,18). The results of double-staining showed that most tubulesstaining for LIF expressed AQP-1, however, some AQP-1–negativetubules contained LIF-positive cells (Figure 5C). This suggeststhat the LIF protein was predominantly localized in the S3 segmentsof the proximal tubules of ischemic kidneys and to some extentin the distal tubules in their outer medulla.
Figure 5. Immunohistochemical localization for LIF and LIF receptor protein in ischemia/reperfusion-injured kidneys 24 h after reperfusion. (A and B) Immunohistochemical localization for LIF protein (brown staining) in the outer medulla of ischemic kidneys. The dramatic increase in LIF immunostaining in injured epithelial tubular cells, especially of S3 segment of the proximal tubules. Detached cells and attached cells in the tubules were stained. (C) Double immunohistochemistry for LIF and aquaporin-1 (AQP-1), marker of proximal tubules. Note that LIF (purple staining) was co-localized with AQP-1 (blue staining). (D) Double immunohistochemistry for LIF and BrdU. BrdU were injected with rat 2 h before sacrifice. Most BrdU staining (brown nuclear staining) was found in proximal tubules expressing LIF (purple cytoplasmic staining). (E) Immunohistochemical localization for LIF receptor (brown staining) in the outer medulla of ischemic kidneys. The expression pattern is similar to that of LIF. Magnifications: x100 in A; x400 in B–D; x200 in E.
We then examined the relationship between LIF expression andtubular cell proliferation after I/R injury. Cell proliferationwas assessed by incorporation of BrdU injected into the rats2 h before sacrifice. BrdU-positive cells were rarely observedin normal kidney. In ischemic kidney, BrdU-positive cells wereobserved predominantly in the outer medulla, where damage tothe tubule cells was most obvious. Double immunohistochemicalstaining revealed that most BrdU-positive cells were presentin tubules expressing LIF (Figure 5D).
LIFR was also predominantly localized in the proximal tubularcells of the outer medulla in the I/R-injured kidneys, coincidingwith the distribution of LIF protein (Figure 5E).
Expression of LIF mRNA in an In Vitro Model of Ischemic Renal Injury
An in vitro model of ischemic renal injury in cultured rat renalepithelial cell line NRK 52E (19) was used to further elucidatewhether LIF is involved in renal tubular regeneration. The invitro model of ischemic renal injury was created by inducingATP depletion by producing chemical anoxia with the mitochondrialinhibitor antimycin A. When monolayers of NRK 52E cells wereexposed to 1 µM antimycin A, cellular levels of ATP rapidlydeclined to 2.1% of the control values within 30 min (Figure 6).This effect could be reversed by removing the antimycinA and restoring the regular medium. The ATP level recoveredto 64.0% of the control values 3 h after ATP repletion.
Figure 6. Cellular ATP levels after chemical anoxia using antimycin A in NRK 52E cells. Confluent NRK 52E cells were incubated in Dulbecco PBS with 1.5 mM CaCl2, 2 mM MgCl2, and either 1 µM antimycin A. Intracellular ATP levels were measured by a luciferase-based assay at the indicated time after ATP depletion. Values are expressed as a percentage of the value of pre-injury and represent means ± SEM from three separate experiments.
The change in LIF mRNA expression during the recovery periodwas quantified by using the real-time PCR method (Figure 7).The LIF mRNA level began to increase on 6 h and persisted until12 h after ATP depletion and then gradually decreased to baseline(Figure 7).
Figure 7. Expression levels of mRNA for LIF in NRK 52E cells after ATP depletion. Total RNA was extracted from cells at the indicated times after ATP depletion. Quantification of mRNA was determined by real-time PCR. Expression levels were normalized to an endogenous control 18S ribosomal RNA (rRNA) and are shown as ratio to the value of 0 h. Values represent means ± SEM for four independent experiments.
Role of LIF in Tubular Regeneration after ATP Depletion
To investigate the mitogenic effect of LIF on renal tubularcells, we assessed the effect of anti-LIF neutralizing antibodyon recovery from the antimycin A-induced injury. NRK 52E cellswere injured by exposure to antimycin A for 1 h and then incubatedin a recovery medium containing anti-LIF neutralizing antibody.The number of surviving cells was determined 48 h after ATPdepletion (Figure 8A). Blocking endogenous LIF protein with1.0 µg/ml of anti-LIF antibody induced a significant reductionin cell number at 48 h during the recovery phase. DNA synthesiswas also measured as incorporation of BrdU. In agreement withthe results for cell number, 1.0 µg/ml of anti-LIF antibodylowered DNA synthesis at 24 h during recovery (Figure 8B). Nosignificant change was observed in NRK 52E cells incubated withnormal goat IgG (data not shown). To determine whether the mitogeniceffect of endogenous LIF was specific to the post-injury period,we performed similar studies with uninjured NRK 52E cells. Incontrast to the results in the post-injury period, anti-LIFantibody had no effect on cell number or DNA synthesis in uninjuredNRK 52E cells (Figure 9).
Figure 8. The effect of anti-LIF neutralizing antibody on recovery from ATP depletion in NRK 52E cells. The injured cells were allowed to recover in regular growth medium containing 0, 0.1, 0.2, or 1.0 µg/ml of anti-LIF antibody. (A) Cell number on day 2 (n = 4) and (B) BrdU incorporation on day 1 (n = 6) after ATP depletion were decreased by 1.0 µg/ml of anti-LIF antibody. The values are indicated as percentage of the value without anti-LIF antibody, and means ± SEM. * P < 0.05 versus the value without anti-LIF antibody.
Figure 9. The effect of anti-LIF neutralizing antibody on proliferation in NRK 52E cells. The non-injured cells were incubated in regular growth medium containing 0, 0.1, 0.2, or 1.0 µg/ml of anti-LIF antibody. (A) Cell number on day 2 (n = 4) or (B) BrdU incorporation on day 1 (n = 6) were not changed. The values are indicated as percentage of the value without anti-LIF antibody and means ± SEM. NS, no significant difference.
It has been suggested that regeneration processes may recapitulatedevelopmental paradigms to restore organ or tissue function(3,4). Several genes including Pax-2, Wnt-4, and activin-A,which play a pivotal role in nephrogenesis, have been shownto be re-expressed in the kidney in the regeneration processfollowing injury (8–11). We focused our attention on LIF,since it has been shown to play an important role in renal development(16,22). This study demonstrates for the first time that LIFparticipates in kidney regeneration after ischemic injury.
The metanephros, which becomes the permanent kidney, arisesfrom two mesodermal derivatives: the ureteric bud and the metanephricmesenchyme. Once the ureteric bud grows out from the Wolffianduct and encounters the mesenchyme, a series of reciprocal inductiveevents takes place that results in the ureteric bud growingand branching to form the collecting ducts, and the metanephricmesenchyme condensing and forming the tubules and the glomeruli(23). LIF secreted by the ureteric buds plays an important rolein the conversion of the mesenchyme into epithelium (16,22);however, the spatiotemporal expression patterns of LIF and itsreceptor have not been fully elucidated. Accordingly, we firstinvestigated the localization of LIF and LIFR in fetal and adultrat kidney.
In developing rat kidney, expression of LIF and LIFR was observedin the derivatives of the ureteric buds on E17 and thereafter.As nephrogenesis progressed, the ureteric buds differentiatedinto collecting ducts, which consistently expressed LIF andLIFR even after nephrogenesis had been completed. Only LIFRwas expressed in the mesenchyme, with no expression of LIF inmesenchymal cells being detected in either fetal or adult kidney.LIFR expression in the mesenchymal cells was already apparenton E15, and it was sustained until the neonatal kidney.
The nephrogenic zone of the embryonic kidney is where primitivetubules and glomeruli are generated. In the nephrogenic zone,the tips of ureteric buds were observed to express LIF and LIFR,whereas the surrounding mesenchymal cells expressed LIFR. Presumably,secreted LIF binds to the LIFR located in the ureteric budsin an autocrine fashion and to the LIFR located in mesenchymein a paracrine fashion. The LIF-LIFR system is speculated toplay a pivotal role in nephrogenesis, and that would be consistentwith the results reported by other investigators (16,22). Inthe adult kidney, expression of LIF and LIFR was most oftenlocalized in the collecting ducts. These changes in distributionsuggest a different role of LIF/LIFR in the embryonic and adultkidney.
LIF is a pleiotropic cytokine that is particularly involvedin growth and development. In the kidney, it is known to playan important role in nephrogenesis. In the adult, LIF has beenshown to be involved in a variety of acute and chronic inflammations.LIF has been identified within the urine of renal allograftrecipients during episodes of acute rejection (24). The LIFexpression level in cultured mesangial cells has been shownto be increased by cytokines (25). Glomerulonephritis inducesLIF in the glomeruli, and administration of exogenous LIF amelioratesglomerulonephritis (26). The function of LIF in the kidney underphysiologic conditions is largely unknown, although there isa solitary report by Tomida M et al. (27) on the action of LIFon renal tubule cells. They found that LIF inhibits the developmentof Na+–dependent hexose transport in LLC-PK1 cell, whichwas isolated from pig kidney and is thought to have the characteristicsof proximal tubule cells. In our study, LIF was found to bepredominantly expressed in the collecting duct cells. The findingsof Tomida et al. (27) and our own suggest that LIF may be involvedin ion transport in renal collecting ducts.
Next, we tested the hypothesis that LIF is involved in the processof kidney regeneration after injury by investigating expressionof LIF and LIFR during the recovery phase from injury by real-timePCR, in situ hybridization, and immunohistochemistry in a modelof acute renal failure created by inducing ischemia. Our firstfinding in the ischemic kidneys was that the increase in LIFmRNA expression began on day 1 after the injury and was sustainedfor 7 d, whereas the increase in LIFR mRNA expression was observedseveral days later. Many genes have been reported to be upregulatedafter tubular injury. Egr-1 (28), c-fos (28), c-myc (29), andthe genes encoding heat shock protein-70 (30) and parathyroidhormone–related protein (PTHrP) (31) have been shown tobe upregulated in the very early phase after injury. Their upregulationwas detected as early as within a few hours and returned tobaseline within 1 d. These are considered the early responsegenes to renal injury. The increase in gene expression of Pax-2(8,11), activin-A (10), platelet-derived growth factor (PDGF)(32), ciliary neurotrophic factor (CNTF) (33), hepatocyte growthfactor receptor (c-Met) (34), galectin-3 (35), and transforminggrowth factor–1 (TGF-1) (36) persisted for several days,and these genes are thought to be responsible for tubular regeneration.The upregulation of LIF was observed on 1 d after the ischemicinsult and persisted for 7 d. Its time course suggests thatLIF participates in the regeneration process. Interestingly,there was a time lag between the gene upregulation of LIF andLIFR, and there is a possibility that LIF itself or some othermolecules in the downstream of LIF signaling may stimulate thetranscription of LIFR.
We did not perfuse the kidney to remove the blood before totalRNA isolation. The possibility of contamination by RNA fromblood cells cannot be ruled out, however, any contribution byblood cells to the increase in LIF mRNA is thought to be minimal.The increase in LIF expression is mainly attributable to theregenerating epithelial tubular cells, and this is supportedby two observations. First, immunohistochemistry and in situhybridization studies showed the most prominent staining ofLIF in the epithelial cells. Second, in vitro study in the absenceof blood cells demonstrated upregulation of LIF mRNA.
Our second observation in the ischemic kidneys was that theincrease in expression of LIF and LIFR was predominantly inthe outer medulla, especially in the S3 segment of the proximaltubules, where LIF or LIFR is not usually expressed. The outermedulla is also the most prominent site of ischemia-inducedacute tubular necrosis and subsequent DNA synthesis (29,37).In our study, most of the cells that stained with BrdU werefound in LIF/LIFR-positive tubules. Interestingly, LIF and LIFRwere expressed in the detached cells as well as the attachedcells of S3 segment of the proximal tubules, and in previousstudies, c-Met (34), galectin-3 (35), TGF-1 (36), and PDGF (32)were shown to be expressed strongly in the detached cells ofS3 proximal tubules. By the same analogy as in the speculationsabout these genes, the increase in LIF staining in the detachedcells presumably reflects an unsuccessful protective responseto injury before the cells’ detachment and necrosis.
Although LIF expression is restricted to the ureteric bud andits derivatives, the major site of action of LIF is the mesenchyme.LIF is excreted by the ureteric bud and induces a mesenchymalto epithelial conversion via binding to its receptor locatedin the mesenchyme. Thus, LIF is important to the developmentof metanephric mesenchyme-derived structures. In view of this,the involvement of LIF in regeneration of the injured proximaltubules, which are derived from the mesenchyme, is consistentwith the functional role of LIF in developing kidney. However,we do not know why LIF is expressed in bud-derived structuresduring development and re-expressed in mesenchyme-derived structuresin disease. The expression pattern of galectin-3 is similarto that of LIF. Galectin-3 is expressed in ureteric bud-derivedstructures in fetal and adult kidney and re-expressed in injuredproximal tubule cells (35,38). One hypothesis to explain there-expression in cells of different origin in disease is thatthe regenerating proximal tubule cells are dedifferentiatedand have different characteristics.
LIF is a member of the IL-6 family, which also includes IL-6,IL-11, oncostatin M, cardiotrophin-1, and CNTF. IL-6 familymembers share the same intracellular signaling system, gp130/JAK/STAT(39). To our knowledge, there have been no reports on involvementof LIF in epithelial cell regeneration. However, two membersof the IL-6 family, IL-6 and CNTF, have been shown to participatein renal tubule cell regeneration after kidney injury. Administrationof IL-6 stimulates tubule regeneration after glycerol-inducedacute renal failure (40), and CNTF has been shown to be involvedin renal tubule regeneration after ischemia-reperfusion injury(33). These observations suggest the importance of the roleof the IL-6 family in renal tubule cell regeneration. LIF itselfhas been shown to prompt the regeneration processes after damageto other cells and organs, including neurons (41), the liver(42), heart (43), and muscle (44). In this study, we showedthat LIF expression is upregulated after an ischemic insultand that its expression is localized in BrdU-positive, proliferatingproximal tubules. These findings provide evidence in supportof our hypothesis that LIF participates in renal tubule regenerationafter renal injury.
To clarify the role of LIF in the regeneration of tubular cells,we utilized the reversible in vitro model of ATP depletion inNRK 52E cells. The cell injury caused by ATP depletion is thoughtto mimic the effect of ischemia in vivo, and the recovery phaseafter ATP depletion is thought to reproduce the regeneration(45). With this in mind, we used anti-LIF neutralizing antibodyto examine the role of LIF in the mitogenic response duringthe recovery phase from injury. The blocking of endogenous LIFwith anti-LIF neutralizing antibody significantly reduced cellnumber and DNA synthesis after recovery from ATP depletion,and the mitogenic effect of endogenous LIF appeared to be specificto the post-injury period, since anti-LIF antibody had no effecton non-injured NRE 52E cells. Tubular cell proliferation isthe hallmark of early regeneration after ischemic renal injury(29), and the results of the in vitro study provide strong supportfor the hypothesis that LIF plays a pivotal role in renal epithelialregeneration after injury.
In conclusion, we have demonstrated that LIF expression in thekidney is transiently upregulated after an ischemic insult.The greatest increase in LIF occurred in the damaged proximaltubules in the outer medulla, and LIF protein was co-localizedwith the proliferation marker BrdU. The blockade of endogenousLIF also reduced the regeneration after in vitro injury. Onthe basis of these findings as well as our observations in thedeveloping kidney, we concluded that the LIF/LIFR axis is reactivatedduring renal regeneration after I/R injury and that it may recapitulatethe developmental process to restore organ or tissue function.
Acknowledgments
This work was supported, in part, by grants from the Ministryof Education, Science and Culture of Japan, Keio UniversityGrant-in-Aid for Encouragement of Young Medical Scientists,and a National Grant-in-Aid for the Establishment of High-TechResearch Center in a Private University.
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Received for publication August 8, 2003.
Accepted for publication September 21, 2003.
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Abstract
Introduction
1 (TGF-
