Jean-Philippe Pradère*,,
Julie Klein,,
Sandra Grès*,,
Charlotte Guigné*,,
Eric Neau,,
Philippe Valet*,,
Denis Calise,
Jerold Chun||,
Jean-Loup Bascands,,
Jean-Sébastien Saulnier-Blache*, and
Joost P. Schanstra,
* Inserm, U858/I2MR, Department of Metabolism and Obesity, Team 3, and Department of Renal and Cardiac Remodeling, Team 5; Université Toulouse III Paul Sabatier, Institut de Médecine Moléculaire de Rangueil; Zootechny Department IFR31, Institut Louis Bugnard, Toulouse, France; || Department of Molecular Biology Helen L. Dorris Child and Adolescent Neuropsychiatric Disorder Institute The Scripps Research Institute, La Jolla, California
Correspondence: Dr. Joost Schanstra, Inserm, U858/I2MR, Department of Renal and Cardiac Remodeling, Team #5, 1 Avenue Jean Poulhès, BP 84225, 31432 Toulouse, Cedex 4, France. Phone: +33-5-6132-3748; Fax: +33-5-6217-2554; E-mail: schans{at}toulouse.inserm.fr
Received for publication February 13, 2007.
Accepted for publication July 6, 2007.
Tubulointerstitial fibrosis in chronic renal disease is stronglyassociated with progressive loss of renal function. We studiedthe potential involvement of lysophosphatidic acid (LPA), agrowth factor–like phospholipid, and its receptors LPA1–4in the development of tubulointerstitial fibrosis (TIF). Renalfibrosis was induced in mice by unilateral ureteral obstruction(UUO) for up to 8 d, and kidney explants were prepared fromthe distal poles to measure LPA release into conditioned media.After obstruction, the extracellular release of LPA increasedapproximately 3-fold. Real-time reverse transcription PCR (RT-PCR)analysis demonstrated significant upregulation in the expressionof the LPA1 receptor subtype, downregulation of LPA3, and nochange of LPA2 or LPA4. TIF was significantly attenuated inLPA1 (–/–) mice compared to wild-type littermates,as measured by expression of collagen III, -smooth muscle actin(-SMA), and F4/80. Furthermore, treatment of wild-type micewith the LPA1 antagonist Ki16425 similarly reduced fibrosisand significantly attenuated renal expression of the profibroticcytokines connective tissue growth factor (CTGF) and transforminggrowth factor (TGF). In vitro, LPA induced a rapid, dose-dependentincrease in CTGF expression that was inhibited by Ki16425. Inconclusion, LPA, likely acting through LPA1, is involved inobstruction-induced TIF. Therefore, the LPA1 receptor mightbe a pharmaceutical target to treat renal fibrosis.
The incidence of chronic kidney disease leading to end-stagerenal disease (ESRD) continues to increase throughout the world.1Almost all forms of ESRD are preceded by the progressive appearanceof renal fibrosis (i.e., extracellular matrix (ECM) accumulation).The presence of fibrosis in the tubulointerstitium (i.e., TIF),compared with glomerular sclerosis, correlates strongly withevolution toward ESRD.1,2 The development of TIF can be schematicallydivided: (1) Inflammation associated with infiltration of macrophages,lymphocytes, and an increase in circulating cytokines and chemokines.(2) This inflammation induces disequilibrium between apoptosisand proliferation of tubular cells, as well as accumulationof myofibroblasts. Myofibroblasts infiltrate from the circulationinto the interstitum, appear by epithelial mesenchymal transition(EMT), or appear by proliferation/activation of the few residentfibroblasts. (3) These myofibroblasts are the main cell typeresponsible for the secretion of the ECM.1,3 As these eventsoccur, the amount of fibrotic tissue increases, causing a steadydecline of renal function until eventually the kidney is nolonger able to function and organ failure occurs.
In the past, a number of mediators of TIF development have beenidentified, including chemokines, cytokines, and growth factors.4Among these, TGF is thought to be the most fibrogenic, directlyor indirectly through the action of CTGF.5
LPA is a growth factor–like phospholipid known to regulateseveral cellular processes including motility, proliferation,survival, and differentiation by acting via specific G-protein–coupledreceptors (LPA1, LPA2, LPA3, and LPA4).6 Until now, a limitednumber of pharmacological tools specifically targeting LPA receptorsubtypes have been developed. Among them is the antagonist Ki16425,which has been demonstrated to specifically block LPA1 and LPA3receptor subtypes in vitro.7 Recently, the in vivo efficacyof Ki16425 in blocking the action of the LPA1 receptor subtypehas been demonstrated.8 LPA has been associated with the etiologyof a growing number of disorders,9 but the involvement of LPAin the progression to ESRD is unclear. In acute renal disease,contradictory results were obtained since intraperitoneal injectionof LPA was reported to prevent renal ischemia-reperfusion injury,10whereas pharmacologic blockade of LPA3 receptor was reportedto reduce renal ischemia-reperfusion injury.11 However, in patientswith chronic renal failure, it has been reported that LPA concentrationsare increased.12,13 These observations led us to hypothesizethat LPA could be involved in the response of the kidney toinjuries and could thus contribute to the progression of chronicrenal disease.
The objective of our study was to clearly determine the contributionof LPA in the development of TIF, a hallmark of progressiverenal disease. We studied LPA production and the expressionof LPA receptor subtypes in kidneys subjected to UUO, an acceleratedmodel of TIF.3,14 We observed that UUO-induced renal TIF isaccompanied by an upregulation of LPA1 receptor expression andby an increased release of LPA by the obstructed kidney, UUO-inducedfibrosis is significantly attenuated in kidneys from LPA1(–/–)mice as well as in mice treated with the LPA1 receptor antagonistKi16425, and LPA increases the expression and release of theprofibrotic cytokine CTGF by proximal tubular cells in vitro.These observations argue strongly for the involvement of LPAin the development of renal TIF and lead us to propose thatthe LPA1 receptor may represent an interesting potential therapeutictarget for the treatment renal fibrosis.
UUO-Induced TIF Is Associated with an Increased Release of LPA by Kidney
To determine the possible involvement of LPA in renal TIF, LPAwas quantified in conditioned media prepared from kidney explantsfrom mice at different time points after UUO. The inductionof renal TIF was validated by the increase in the level of mRNAencoding two previously characterized TIF and macrophage markers(collagen III and F4/80, respectively) (Figure 1A).15
Figure 1. Effect of UUO on the release of LPA and the expression of LPA receptor subtypes in the kidney. Mice were subjected to UUO and kidneys were removed 0, 3, 5, and 8 days after surgery. RNA were extracted from total kidneys and mRNA encoding type III collagen (collagen III) and F4/80 (A) and LPA1, LPA2, LPA3, and LPA4 receptor subtypes (C) were quantified by real-time PCR. (B) Explants from operated (UUO) and contralateral nonoperated (cont) kidneys were maintained in primary culture for 6 h, and LPA released in the conditioned medium was quantified by a radioenzymatic assay. Values are means ± SEM from 4 (A through C) and 5 (B) mice for each time point. Comparisons with day 0 were performed using Student t test. *P < 0.05; **P < 0.01.
LPA was present in conditioned media from kidney explants obtainedfrom nonobstructed kidneys (Figure 1B; time 0). When comparedwith time 0, LPA concentration in conditioned media was significantlyhigher at each time point after UUO (3.3-, 3.6-, and 2.9-foldat days 3, 5, and 8, respectively) (Figure 1B). Controlateralkidneys exhibited no significant change in LPA release whencompared with time 0 (Figure 1B). Similarly, sham-operated kidneysexhibited no significant change in LPA release when comparedwith nonoperated mice (data not shown).
UUO-Induced Renal TIF Is Associated with Upregulation of Renal LPA1 Receptor Expression
Four LPA receptor subtypes have been identified (LPA1, LPA2,LPA3, and LPA4).6 Real-time reverse-transcription PCR (RT-PCR)analysis revealed that the four subtypes were expressed in totalkidney extracts from control mice with the following rank order:LPA2 > LPA3 = LPA1 > LPA4 (Table 1). Analysis of LPA receptorsubtype expression separately in the kidney cortex or in thekidney medulla did not change this expression order (Table 1).
Table 1. Expression of LPA-Receptor Subtypes in Kidneya
When compared with time 0, the expression of the LPA1 receptorsubtype was significantly increased at day 5 (2.8-fold) andday 8 (4.8-fold) after UUO (Figure 1C). In contrast, the expressionof the LPA3 receptor was significantly decreased at day 3 (4-fold),day 5 (3-fold), and day 8 (4.5-fold) when compared with time0. No significant change in LPA2 and LPA4 receptor expressionwas observed (Figure 1C). Eight days after surgery, controlateraland sham-operated kidneys exhibited no significant change ingene expression when compared with time 0 (data not shown).
Attenuation of UUO-Induced Renal TIF in LPA1 Receptor Knockout Mice
The above data suggested that LPA could play a role in UUO-inducedrenal fibrosis via the activation of the LPA1 receptor. To testthis hypothesis, the level of UUO-induced renal TIF was comparedbetween LPA1(–/–)16,17 and LPA1(+/+) mice. LPA1(–/–)mice exhibited a slight but nonsignificant reduction in LPA2receptor expression when compared with LPA1(+/+) mice (Table 2).No significant change was observed for LPA3 receptor mRNA expression.In LPA1(+/+) mice with a mixed 129SvJ/C57BL/6J background, basalLPA1 receptor expression was lower than in mice with a pureC57BL/6J background. The LPA4 receptor was not detectable inmice with the mixed genetic background (Table 2). As shown inFigure 2, mRNA expression of typical fibrosis markers such ascollagen type III, -smooth muscle actin (SMA), which is a markerof tubulointerstitial myofibroblasts responsible for a largecomponent of collagen deposition in the interstitium, or F4/80(inflammation) was significantly lower in LPA1(–/–)than in LPA1(+/+) mice. This was confirmed at the protein levelfor collagen type III and SMA (Figure 3, A and B). Inductionof F4/80 protein tended to be lower in LPA1(–/–)versus LPA1(+/+) mice, but the difference did not reach statisticalsignificance (Figure 3C).
Figure 2. Influence of LPA1-receptor gene knockout on UUO-induced renal TIF (mRNA expression). LPA1-receptor knockout mice (–/–) and their wild-type (+/+) littermates were subjected (black bars) or not (white bars) to UUO; kidneys were removed 8 d after surgery. mRNA expression was quantified by real-time PCR: (A) collagen III; (B) -smooth muscle actin (SMA); (C) F4/80. Values are means ± SEM from 6 mice by group. Amplitudes of UUO-induced fibrosis between (+/+) and (–/–) mice were compared by using a two-way ANOVA test. *P < 0.05.
Figure 3. Influence of LPA1-receptor gene knockout on UUO-induced renal TIF (protein expression). LPA1-receptor knockout mice (–/–) and their wild-type (+/+) littermates were subjected to UUO (black bars) or not (white bars). Kidneys were removed 8 d after surgery, and protein expression was analyzed with immunohistochemistry: (A) type III collagen; (B) SMA; (C) F4/80. Representative photographs are shown on the left. Quantification of the photographs is shown on the right. Values are means ± SEM of 6 mice by group. Amplitudes of UUO-induced fibrosis between (+/+) and (–/–) mice were compared by two-way ANOVA test. *P < 0.05. Calibration bar, 250 µm.
Attenuation of UUO-Induced Renal TIF by Ki16425 Treatment
Attenuation of UUO-induced TIF in LPA1(–/–) micestrongly suggested that the LPA1 receptor was involved in thedevelopment of TIF. To strengthen this hypothesis we performeda pharmacological knockout of the LPA1 receptor by treatingobstructed mice with the LPA1 receptor antagonist Ki16425.7,8In nonobstructed mice, Ki16425 treatment did not significantlychange the renal expression of the LPA1, LPA2, and LPA4 receptorswhen compared with vehicle-treated mice. A slight but nonsignificantincrease in LPA3 receptor expression was observed (Table 2).UUO-induced fibrosis (collagen type III, SMA) and inflammatory(F4/80) mRNA markers were significantly lower in Ki16425-treatedmice than in control mice (Figure 4). This was confirmed atthe protein level for F4/80 and collagen type III (Figure 5).
Figure 4. Effect of Ki16425 treatment on UUO-induced renal TIF (mRNA expression). Mice were subjected to UUO (black bars) or not (white bars) in combination with a daily injection of Ki16425 or its vehicle. Kidneys were removed 8 d after surgery, and mRNA expression was determined by real-time PCR: (A) type III collagen; (B) SMA; (C) F4/80. Values are means ± SEM from 6 mice by group. Amplitudes of UUO-induced fibrosis between vehicle- and Ki16425-treated animals were compared by two-way ANOVA test. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 5. Effect of Ki16425 treatment on UUO-induced renal TIF (protein expression). Mice were subjected to UUO (black bars) or not (white bars) in combination with a daily injection of Ki16425 or its vehicle. Kidneys were removed 8 d after surgery, and protein expression was analyzed by immunohistochemistry: (A) F4/80; (B) type III collagen. Representative photographs are shown on the left. Quantification of the photographs is shown on the right. Values are means ± SEM from 6 mice by group. Amplitudes of UUO-induced fibrosis between vehicle- and Ki16425-treated animals were compared by two-way ANOVA test. *P < 0.05; **P < 0.01. Calibration bar, 250 µm.
Effect of LPA on CTGF and TGF Expression In Vivo
CTGF was previously demonstrated to play a crucial role in UUO-inducedTIF,18,19 and was involved in the profibrotic action of TGF.5We therefore analyzed TGF and CTGF mRNA expression in obstructedmice treated with the LPA receptor antagonist Ki16425. We observedthat Ki16425 treatment led to a strong attenuation (3- to 4-fold)in the induction of TGF and CTGF mRNA expression by UUO (Figure 6).These data suggested the involvement of TGF and CTGF in theprofibrotic action of LPA.
Figure 6. Effect of Ki16425 treatment on UUO-induced renal TIF: Expression of profibrotic cytokines. Mice were subjected to UUO (black bars) or not (white bars) in combination with a daily injection of Ki16425 or its vehicle. Kidneys were removed at day 8 after surgery, and mRNA expression was determined by real-time PCR: (A) TGF; (B) CTGF. Values are means ± SEM from 6 mice by group. Amplitudes of UUO-induced fibrosis between vehicle- and Ki16425-treated animals were compared by two-way ANOVA test. **P < 0.01.
Effect of LPA on CTGF and TGF Expression In Vitro
Finally, we tested whether the profibrotic action of LPA couldresult from a direct impact of LPA on kidney cells. For that,the mouse epithelial renal cell line MCT was treated with LPA.20Real-time RT-PCR analysis revealed that MCT cells mainly expressedLPA1 and LPA2 receptor subtypes (ratios of 28 ± 7 and21 ± 3 to 18S RNA(x10,000), respectively), whereas LPA3and LPA4 receptor subtypes were undetectable. LPA induced arapid and transient (Figure 7A) and a dose-dependent (Figure 7B)increase (10-fold maximum) in CTGF mRNA expression. In parallel,LPA exerted only a weak but significant increase (3-fold after6 h) on TGF mRNA expression (Figure 7, A and B). CTGF mRNA inductionby LPA was almost completely suppressed by cotreatment withthe LPA-receptor antagonist Ki16425 (Figure 7C). LPA treatmentwas also accompanied by a release of CTGF protein in the culturemedium of MCT cells, and that release was suppressed by cotreatmentwith Ki16425 (Figure 7D).
Figure 7. Effect of LPA on CTGF expression in MCT cells. CTGF and TGF mRNA were quantified in serum-starved MCT cells exposed to 2 µM LPA for increasing time (A) or to increasing concentrations of LPA for 2 h (B); ***P < 0.001 when compared with time 0 (A) or to the absence of LPA (B) (determined by t test). (C) CTGF mRNA were quantified in serum-starved MCT cells exposed to 2 µM LPA ± 10 µM Ki16425: *P < 0.05; **P < 0.01 when compared with LPA alone (determined by t test). Values are means ± SEM from 3 separate experiments. (D) Serum-starved MCT cells were exposed to 2 µM LPA ± 10 µM Ki16425, and the release of CTGF protein in the culture medium for 3 h was analyzed by Western blot (representative of 2 separate experiments).
This study shows that (1) UUO-induced renal TIF is accompaniedby an increased release of LPA, and by an upregulation of LPA1receptor expression in the obstructed kidneys; (2) UUO-inducedfibrosis is significantly attenuated in kidneys from LPA1(–/–)mice as well as in mice treated with the LPA1 receptor antagonistKi16425; and (3) on renal proximal tubular cells in vitro, LPAincreases the expression and release of the profibrotic cytokineCTGF. These observations strongly argue for the involvementof LPA in the development of renal TIF and lead us to proposethat the LPA1 receptor may represent an interesting pharmaceuticaltarget for the treatment of chronic renal disease.
The metabolic origin of LPA released by the kidney, as wellas the mechanisms by which the release of LPA is increased afterUUO, remain unknown. Several enzymes, including phospholipasesA1/A2, lysophospholipase D/autotaxin, glycerol-phosphate acyltransferase,or monoacylglycerol kinase, can possibly lead to renal synthesisof LPA.21 Expression and/or the activity of one of these enzymesmight be increased in the kidney as an adaptive response tochronic kidney injury induced by UUO. In rat, UUO was shownto increase the activity of a phosphoethanolamine-specific phospholipaseA2.22 The involvement of this enzyme in LPA synthesis in theobstructed kidney remains to be explored.
LPA is a growth factor–like phospholipid known to regulateseveral cellular processes via the activation of specific G-protein–coupledreceptors (LPA1, LPA2, LPA3, and LPA4).6 We observed that UUOsignificantly upregulated LPA1 receptor expression, which suggeststhat this subtype may play an important role in UUO-inducedfibrosis. This hypothesis is supported by our results showingthat UUO-induced TIF is significantly attenuated in LPA1 receptorknockout mice, as well as in mice treated with the LPA1 receptorantagonist Ki16425. Nevertheless, we found that kidneys alsoexpress LPA2 and LPA3 receptor subtypes, confirming previousreports,11,23 and that UUO reduced LPA3 receptor expression.Therefore, the involvement of LPA2 and LPA3 receptor subtypesin the action of LPA in the development of renal TIF cannotbe excluded. In the future, the development of specific LPA2or LPA3 receptor antagonists may help address that hypothesis.
Currently it is not known which renal cells are specificallytargeted by LPA and which cells are involved in the LPA1 receptor–mediatedrenal fibrosis in ureteral obstruction. The development of renalTIF in UUO is associated with infiltration of inflammatory cells,transformation of epithelial cells into myofibroblasts, proliferationof (myo)fibroblasts, tubular atrophy, and secretion of ECM.On the basis of the literature, LPA can potentially regulatesome of these events. LPA has, for example, been demonstratedto participate in intraperitoneal accumulation of monocytes/macrophages24,25as well as in the control of the proliferation of nonrenal myofibroblasts26and mesangial cells via the activation of the ras/MAPK pathway.27On the basis of our results and previous reports,11,23 the expressionof the LPA1 receptor is not different between renal cortex andmedulla, suggesting that this receptor subtype is ubiquitouslyexpressed throughout the different areas of the kidney. Consequently,the kidney cell type that is preferentially involved in theprofibrotic activity of LPA remains to be defined. However,on the basis of the observation that UUO-induced fibrosis isessentially interstitial, without visible glomerular lesions,14,28the glomerular LPA1 receptor is most likely not involved inthe effects of LPA on UUO-induced TIF. The remaining cell typesthat can be potential targets of LPA in the development of UUO-inducedrenal fibrosis therefore include tubular and inflammatory cellsand interstitial fibroblasts. Because LPA was already knownto participate in intraperitoneal accumulation of monocytes/macrophages24,25and that LPA can induce expression of the profibrotic cytokineCTGF in primary culture human fibroblasts,29 we focused theremainder of our studies on the in vitro effects of LPA treatmenton tubular cells. In addition, it has been shown that primaryculture human proximal tubular cells express the LPA1 receptor.30
Among the UUO-induced factors that are strongly attenuated byLPA1 receptor blockade is the profibrogenic factor CTGF. Interestingly,we found that LPA was able to upregulate CTGF expression andsecretion in cultured proximal tubular cells. Similar observationswere made previously in renal fibroblasts and mesangial cells.29,31,32Our results show that the action of LPA on CTGF expression isvery likely mediated by the LPA1 receptor subtype because Ki16425blocks these effects. Consequently, the parallel between invivo and in vitro experiments suggests that the profibrogeniceffect of LPA could in part be mediated by increased CTGF expressionand secretion. CTGF induction by LPA in mesangial cells wasshown to be mediated by the small GTPase rhoA and the downstreamkinase ROCK.31 Interestingly, treatments with ROCK inhibitorshave been described to attenuate UUO-induced renal TIF,33 similarto what we observed in LPA1(–/–)- and in Ki16425-treatedmice.
The in vivo expression of the profibrogenic factor TGF is alsosignificantly attenuated by LPA1 receptor blockade. In contrastto CTGF, in vitro LPA treatment of MCT cells only modestly modifiedTGF expression. This difference suggests that regulation ofTGF and CTGF expression and secretion by LPA involves differenttransduction pathways and/or can occur in different kidney celltypes.
Therefore, combining our studies and the published data on theeffects of LPA on renal CTGF and TGF production, the antifibroticeffect of LPA1 receptor blockade can potentially involve threecell types with important roles in the development of UUO-inducedTIF: inflammatory cells, tubular cells, and fibroblasts.
In conclusion, our study demonstrates for the first time, usingboth genetically engineered animals and pharmacological tools,that LPA and its LPA1 receptor could play an important fibroticrole in UUO-induced TIF via a mechanism involving in part theprofibrotic cytokine CTGF. Because TGF has many other effects,34its blockage is not a realistic therapeutic option to reducerenal fibrosis. On the other hand targeting the CTGF has beenshown as a promising antifibrotic therapy.19 Therefore, pharmacologicalinhibition of LPA synthesis or antagonizing LPA1 receptors mightbe interesting in the treatment of renal fibrosis.
Animals
Male LPA1(–/–) mice and their wild-type (WT) littermateswere on a mixed 129SvJ/C57BL/6J background.16,17 For all otherexperiments, C57BL/6J mice were used (Harlan, Gannat, France).Mice were handled in accordance with the principles and guidelinesestablished by the National Institute of Medical Research (INSERM).They were housed in a pathogen-free animal facility with constanttemperature (20 to 22°C), humidity (50 to 60%), and witha 12-h/12-h light/dark cycle (lights on at 8:00 a.m.). All micehad free access to food (energy contents in % kcal: 20% protein,60% carbohydrate, and 20% fat; (Usine d'Alimentation Rationelle,Villemoisson-sur-Orge, France) and water throughout the experiment.
UUO
Mice (8 wk old) were used in these experiments. UUO was performedas described previously.15 Mice were euthanized at differenttime points (0, 3, 5, and 8 d) after UUO, and the kidneys weredissected for further analysis. Control kidneys were dissectedfrom nonoperated mice. All experiments reported were conductedin accordance with the principles and guide lines establishedby INSERM and were approved by a local animal care and use committee.
Treatment with Ki16425
Ki16425 (Sigma, Saint Quentin Fallavier, France) powder wasfirst diluted in DMSO at the concentration of 100 µg/µland then in PBS at the final concentration of 5 µg/µl.Male C57BL/6J mice were injected subcutaneously with the Ki16425solution at the dose of 20 mg/kg per d or with the vehicle (100µl injection volume). Injections began 1 d before UUOsurgery and were repeated daily for 8 d.
Culture of Kidney Explants
Explants were prepared from the distal pole of the kidneys.Explants (9 to 30 mg) were incubated at 37°C in 12 wellsper plate containing 1 ml serum-free DMEM supplemented with1% BSA (97% free fatty acids; Sigma) for 6 h in a humidifiedatmosphere containing 7% CO2. After incubation, conditionedmedia were separated from explants, centrifuged to eliminatecell debris, and frozen at –20°C for further analysis.
LPA Quantification
LPA was extracted from conditioned media and quantified by radioenzymaticassay as described previously.35
mRNA Quantification
Total RNAs were extracted using the RNeasy mini kit (QiagenGmbH, Hilden, Germany). Gene expression was analyzed using real-timeRT-PCR as described previously.36 Oligonucleotides for mousegene expression studies were:
Immunohistochemistry
Immunohistological staining and analysis of kidney sectionswere performed as described previously.15 Rat monoclonal antibodyto mouse F4/80 (RM2900; Caltag Laboratories Inc., Burlingame,CA) was used for macrophage detection. Collagen type III and-SMA were detected using rabbit anti-human collagen type III(T59105R Interchim, Montluçon, France) and the monoclonalmouse anti-human -SMA (DAKO EPOS method, U7033; DAKO S.A., Trappes,France), respectively. For the visualization of collagen typeIII, the DAKO Envision System was used (DAKO S.A.). For allsamples, negative controls for the immunohistochemical proceduresincluded substitution of the primary antibody with nonimmunesera.
Histomorphometric Analysis
As described previously,15 an operator unaware of the originof each kidney section performed analyses. Under a light microscope(Nikon Eclipse 600, Tokyo, Japan) at x200 magnification, 10nonoverlapping fields (to obtain approximately 80% of the kidneysection) per kidney section were captured with an analogic camera(MicroFire CCD color; Optronics, Goleta, CA) connected to themicroscope. Quantitative analysis of the pictures was performedwith Adobe PhotoShop 5.5 software (Adobe Systems Incorporated,San Jose, CA), which allows counting of the pixels stained specifically(brown for the immunohistochemical studies).
Culture of MCT Cells and Preparation of Conditioned Media
MCT cells were a kind gift of Dr M. Zeisberg (Harvard MedicalSchool, Cambridge, MA). Cells were grown until confluence inDMEM supplemented with 5% fetal calf serum. MCT cells were washedtwice with PBS to remove serum and then incubated (4 ml fora 10-cm diameter plate) in serum-free DMEM supplemented for3 h with or without pharmacological reagents. Conditioned mediumwas collected and centrifuged to eliminate cell debris, andconcentrated (about 50 fold) using an Amicon Ultra 10,000 (Millipore)and stored at –20°C before analysis.
Detection of CTGF Secretion by Western Blot
Concentrated conditioned medium (50 µg) were loaded andseparated on a Gel Nu-PAGE (Invitrogen, Cergy Pointoise, France)4-20% and transferred on nitrocellulose membrane. The blot wasincubated overnight at 4°C in TBS/Tween 0.1% containing5% BSA and then for 1 h at room temperature in the same solutionsupplemented with 0.4 µg/ml CTGF antibody (Santa CruzBiotechnology, Santa Cruz, CA). After washing in TBS/Tween 0.1%,CTGF was visualized by enhanced chemoluminescence detectionsystem using an anti-rabbit–horseradish peroxidase antibody.
Statistical Analysis
Values are means ± SEM. The interaction of UUO-inducedfibrosis with LPA1 knockout or Ki16425 treatment was statisticallyanalyzed by a multivariate analysis (two-way ANOVA). Other comparisonswere performed with a t test. Differences were considered significantat P < 0.05.
This work was supported by grants from INSERM and the NationalInstitutes of Health (MH51699 and NS048478). J.K. was supportedby a grant from the Ministère de l'Education Nationalede la Recherche et de la Technologie (France). We thank Y. Barreiraand C. Nevoit (IFR31 Animal Facility), J.J. Maoret (IFR31 MolecularBiology Platform) for technological assistance. We would liketo thank Dr M. Zeisberg for his generous gift of MCT cells.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Abstract
Introduction
-smooth muscle actin (
(TGF
97% free fatty acids; Sigma) for 6 h in a humidified atmosphere containing 7% CO2. After incubation, conditioned media were separated from explants, centrifuged to eliminate cell debris, and frozen at –20°C for further analysis. 
