Deletion of mineralocorticoid receptors in smoothmuscle cells blunts renal vascular resistance followingacute cyclosporine administrationCristian A. Amador1,8, Jean-Philippe Bertocchio1,2,8, Gwennan Andre-Gregoire1, Sandrine Placier3,Jean-Paul Duong Van Huyen4, Soumaya El Moghrabi1, Stefan Berger5, David G. Warnock6,Christos Chatziantoniou3, Iris Z. Jaffe7, Philippe Rieu2 and Frederic Jaisser1
1INSERM UMRS 1138, Team 1, Research Centre of Cordeliers, Paris, France; 2Nephrology, Dialysis and Transplantation Unit, ReimsUniversity Hospital, Reims, France; 3Hemodynamic Platform, Tenon Hospital, Paris, France; 4Pathology Department, Necker Hospital,Paris, France; 5German Cancer Research Center, Heidelberg, Germany; 6University of Alabama at Birmingham, Birmingham, Alabama,USA and 7Molecular Cardiology Research Institute, Tufts Medical Center, Boston, Massachusetts, USA
Calcineurin inhibitors such as cyclosporine A (CsA) are stillcommonly used after renal transplantation, despite CsA–induced nephrotoxicity (CIN), which is partly related tovasoactive mechanisms. The mineralocorticoid receptor (MR)is now recognized as a key player in the control of vasculartone, and both endothelial cell- and vascular smooth musclecell (SMC)-MR modulate the vasoactive responses tovasodilators and vasoconstrictors. Here we tested whethervascular MR is involved in renal hemodynamic changesinduced by CsA. The relative contribution of vascular MR inacute CsA treatment was evaluated using mouse models withtargeted deletion of MR in endothelial cell or SMC. Resultsindicate that MR expressed in SMC, but not in endothelium,contributes to the increase of plasma urea and creatinine, theappearance of isometric tubular vacuolization, andoverexpression of a kidney injury biomarker (neutrophilgelatinase–associated lipocalin) after CsA treatment.Inactivation of MR in SMC blunted CsA–inducedphosphorylation of contractile proteins. Finally, the in vivoincrease of renal vascular resistance induced by CsA wasblunted when MR was deleted from SMC cells, and this wasassociated with decreased L-type Ca2+ channel activity. Thus,our study provides new insights into the role of vascular MRin renal hemodynamics during acute CIN, and providesrationale for clinical studies of MR antagonism to manage theside effects of calcineurin inhibitors.Kidney International advance online publication, 30 September 2015;doi:10.1038/ki.2015.312
Calcineurin inhibitors, such as cyclosporine A (CsA) andtacrolimus, are immunosuppressive drugs widely used afterrenal transplantation. However, calcineurin inhibitors induceacute nephrotoxicity that can result in kidney failure.1
Although the mechanisms underlying CsA–induced nephro-toxicity (CIN) remain unclear,2 alteration of renal hemo-dynamics appears central in acute CIN, and renalvasoconstriction has been reported as an initial event linkedto CIN. CsA promotes renal afferent arteriolar vasoconstric-tion in rats, a pathogenic factor at least as important astubular injury in acute CsA nephrotoxicity.3 In kidneyallograft recipients, daily CsA administration is followed bya transient decrease of the renal blood flow (RBF),4 suggestinga rapid effect of CsA on renal hemodynamics. These effectsare sensitive to angiotensin-converting enzyme inhibitors5
or L-type Ca2+ channel antagonists.4 CsA increasesnoradrenaline-, angiotensin II (AngII)–, endothelin-, andvasopressin-induced vasoconstriction in rat arteries.6–8
Despite these limitations, CsA is still very commonly usedafter organ transplantation, especially in low-incomecountries. Therefore, deciphering the mechanisms by whichCsA promotes renal hemodynamic alterations is crucial to thedevelopment of new therapeutic strategies to prevent orameliorate acute CIN.
Pharmacological blockade of mineralocorticoid receptor(MR) is highly effective in experimental models of CIN.9–11
The beneficial effects of MR antagonism include regulationof renal remodeling (apoptosis, fibrosis) and modulationof vasoactive factors, which are increased during CsAtreatment.12 However, the cellular targets of the MRantagonists (MRAs) and the underlying mechanisms relatedto the vasoconstriction observed in acute CIN are stillunknown. Recent studies have shown that the MR has acentral role in the control of vascular tone: both endothelialcell (Endo)- and vascular smooth muscle cell (SMC)-MRmodulates the vasoactive responses to vasodilators andvasoconstrictors.13–16 The aim of this study was therefore to
Correspondence: Frederic Jaisser, INSERM UMRS 1138, Team 1, ResearchCentre of Cordeliers, 15 rue de l’école de médecine, Paris 75006, France.E-mail: [email protected] first two authors contributed equally to this work.
Received 24 April 2015; revised 13 August 2015; accepted 20 August2015
analyze whether the vascular MR is involved in the renalhemodynamic changes induced by CsA, and if so, to evaluatethe relative contribution of the Endo- or vascular SMC-MR inthis effect.
RESULTSMR gene inactivation in SMC but not in Endo prevents acutekidney failure, tubular vacuolization, and NGAL expressioninduced by acute CsATo determine whether vascular Endo- or SMC-MR is involvedin acute CIN, we administered CsA in Endo-MR-KO(Endo-MR-knockout), SMC-MR-KO, or in control littermate(Ctl) mice. CsA administration induced a similar progressivebody weight loss in Ctl, Endo-MR-KO, and SMC-MR-KOmice (Figure 1a and b). Plasma urea and creatinine wereincreased after 2 days of CsA treatment in Ctl mice(Figure 1c–f) and in Endo-MR-KO mice to a similar extent(Figure 1c and e). However, specific deletion of MR in SMCprevented this increase (Ctl+CsA vs. SMC-MR-KO+CsA:uremia 21.2± 5 vs. 8.1± 0.8mmol/l, creatininemia 33.7± 9.5vs. 10.4± 0.6mmol/l; Po0.05; Figure 1d and f).
CsA–treated Ctl mice developed isometric vacuolization ofthe proximal tubule (Figure 2a–d), similar to the pathologypreviously described in post-transplantation patients withacute CIN17 and in other CIN experimental models.11,18,19
MR deletion in SMC (Figure 2c and d) prevented thedevelopment of these histological lesions, whereas MRdeletion in Endo had no effect (Figure 2a and b).Additionally, immunohistochemistry in renal proximaltubules of Ctl and Endo-MR-KO demonstrated strongexpression of neutrophil gelatinase–associated lipocalin
(NGAL, or Lcn2 in mice), a small glycoprotein used asmarker of tubular injury in mice and humans,20 after 2 daysof CsA treatment (Figure 3a and c). NGAL protein expressionwas induced in Ctl and Endo-MR-KO as determined bywestern blotting of whole kidneys (Figure 3b). MR-KO inSMC prevented the NGAL overexpression induced by CsA(Figure 3d).
MR-KO in SMC prevents CsA–induced phosphorylation ofvascular smooth muscle contractile proteins and modulatesrenal vascular resistance through the activity of L-type Ca2+
channelAs the MR expressed in SMC, but not in endothelial cells, wascritical for acute kidney injury after CsA administration, andas the vasoconstriction has been proposed as a criticalmechanism for acute CIN,2,3,4,11 we explored whether (i) CsAaffected the activation of the endothelial nitric oxide synthase(eNOS) and proteins of the contractile apparatus, and(ii) whether this was modulated by MR deletion in SMC.Activation of eNOS, as measured by phosphorylation ofeNOS at Ser1177, is decreased after CsA treatment to a similarextent in abdominal aortas of Ctl and SMC-MR-KO mice(Figure 4a). Phosphorylation of myosin light-chain kinase(MLCK) at Ser1760 and of myosin regulatory light chain 2(MLC2) at Thr18 and Ser19 are essential for vascular SMCcontraction.21 The phosphorylation levels of MLCK as well asMLC2 proteins were increased in abdominal aortas ofCsA–treated Ctl mice (Figure 4b and c) and Endo-MR-KOmice (Supplementary Figure S1a and 1b online). However,MR deletion in SMC prevented the effect of CsA on both
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Figure 1 |Mineralocorticoid receptor-knockout (MR-KO) in smooth muscle cell (SMC) but not in the endothelium prevents cyclosporineA (CsA)–induced kidney failure. Body weight loss induced by CsA is similar in (a) Endo-MR-KO (endothelial cell-MR-KO) mice and in(b) SMC-MR-KO mice. CsA–induced increase in plasma urea is not prevented in (c) Endo-MR-KO mice, but is prevented in (d) SMC-MR-KO mice.CsA–induced creatinine increase is not prevented in (e) Endo-MR-KO mice, but is prevented in (f) SMC-MR-KO mice. Data are expressed asmean± s.e.m. (n= 6–12). Two-way analysis of variance (ANOVA) test: *Po0.05, **Po0.01, and ***Po0.001 CsA versus VH. Ctl, control;VH, vehicle.
bas i c resea rch CA Amador et al.: Vascular MR in renal hemodynamics after CsA administration
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MLCK and MLC2 phosphorylation (Figure 4b and c), thusaltering a key mechanism in CsA–induced SMC contraction.
Previous clinical observations in transplanted patientsindicated that renal vascular resistance (RVR) is increasedin patients shortly after oral CsA administration.4,22 Toestablish a direct role of SMC-MR in the CsA effects on theintrarenal vasculature, we studied changes of blood pressureand RVR in CsA–treated mice challenged with AngII toinduce a vasoconstrictive response in vivo. Baseline meanarterial blood pressure was similar in Ctl and SMC-MR-KOmice treated with CsA (Ctl+CsA: 81± 3 mmHg;SMC-MR-KO+CsA: 79± 3 mmHg) (Figure 5a). AngIIsimilarly increases mean arterial blood pressure in Ctl andSMC-MR-KO mice (Figure 5b). In vivo administration ofAngII increased RVR in a dose-dependent manner in Ctl mice(Figure 5d). However, the increase in RVR induced by AngIIis blunted in SMC-MR-KO mice compared with Ctl mice(Figure 5c and d).
L-type Ca2+ channels (Cav1.2) are direct targets of MR inthe SMC:15 SMC-MR deletion blunted the activation ofL-type Ca2+ channel in mesenteric arteries.15 As CsA waspreviously shown to increase Ca2+ signaling in the vasculatureby increasing the activity of the membrane Cav1.2 channel,23
we hypothesized that the modulation of Cav1.2 activity inthe renal microvasculature by MR may contribute to the
beneficial effects we observed in SMC-MR-KO mice treatedby CsA. We therefore analyzed the effects of KCl(which allows depolarization-induced L-type Ca2+ channelactivation) and of BayK8644 (a specific L-type Ca2+ channelagonist) on the renal microvasculature perfusion pressure inisolated kidneys after CsA treatment. Renal arteries fromSMC-MR-KO kidneys showed an attenuated contractileresponse to KCl (Figure 5e) and to BayK8644 (Figure 5f)compared with Ctl kidneys. This demonstrated that inSMC-MR-KO mice, the renal vascular activity of the L-typeCa2+ channel is blunted after CsA treatment.
DISCUSSIONOur data indicate that the SMC-MR, but not the Endo-MR, ismandatory for acute kidney failure, tubular vacuolization, andNGAL overexpression induced by CsA administration. Theseeffects are associated to a decreased vasoconstrictive responsein CsA–treated SMC-MR-KO mice that is associated withblunted vascular L-type Ca2+ channel activity.
The alteration in renal hemodynamics has a pivotal role inthe acute nephrotoxicity of transplanted patients treated withCsA as renal vasoconstriction has been established as an initialevent linked to acute CIN.4,5,22 In preclinical models,CsA promotes renal afferent arteriolar vasoconstriction3
and increases the vasoactive response to several
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Figure 2 |Mineralocorticoid receptor-knockout (MR-KO) in smooth muscle cell (SMC) but not in the endothelium prevents cyclosporineA (CsA)–induced tubular vacuolization. CsA induces tubular vacuolization in kidneys from (a) Endo-MR-KO (endothelial cell-MR-KO) mice butnot (c) SMC-MR-KO mice. The scoring of tubular vacuolization for (b) Endo-MR-KO and (d) SMC-MR-KO mice shows that the ablation of MR inSMC blunted the CsA–dependent vacuolization. Bar = 50μm. Data are expressed as mean± s.e.m. (n= 5–9). Two-way analysis of variance(ANOVA) test: *Po0.05 and ***Po0.001 CsA versus VH. Ctl: control; VH: vehicle.
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vasoconstrictors.6–8 Additionally, CsA induces a Ca2+
influx–dependent vasoconstriction in isolated mesentericarteries,23 and increases the perfusion pressure of the caudalartery.24 This arterial vasoconstriction depends on extracel-lular Ca2+.23 It has been proposed that CsA enhances signaltransmission between G-protein-coupled receptors andL-type Ca2+ channel.24 CsA induces Ca2+ influx in vascularSMC through the IP3 pathway by modulating the phosphor-ylation of IP3 receptor.6 Interestingly, the use of Ca2+ channelblockers blunts these effects induced by CsA.23–25 In thepresent study, we showed in CsA–treated mice that SMC-MRdeletion prevents: (1) renal microvascular contraction,(2) phosphorylation of contractile proteins in SMC, and(3) vascular L-type Ca2+ channel activity. Both CsA and theMR expressed in vascular SMC affect the Ca2+ signalingpathway, which is essential for SMC contractility. McCurleyet al.15 recently demonstrated that vascular SMC-MRmodulates the expression of the L-type Ca2+ channel Cav1.2and that SMC-MR deletion blunts the vasoconstrictive effectof a Cav1.2 agonist. In cardiomyocytes, another contractilecell type where MR has an essential role, MR modulates the
activity of both Cav1.2 and the ryanodine receptor, affectingcardiomyocyte contraction.26,27 In the present study, weshowed that the effect of SMC-MR deletion on L-type Ca2+
activity is independent of the abundance of Cav1.2 expression(Supplementary Figure S2a and b online). Furthermore, bymodulating cellular Ca2+ homeostasis, SMC-MR will affectdownstream signaling to the contractile machinery. There-fore, we propose that vascular SMC-MR, by modulatingL-type Ca2+ channel activity and the phosphorylation ofproteins critical for SMC contraction, is a key regulator ofmicrovascular contraction and renal hemodynamics after CsAadministration.
CsA administration has been reported to decrease vascularNO production,28 by increasing the synthesis of superoxideanion in renal endothelial cells,29 and decreasing the activityof eNOS,30 catalase, and glutathione peroxidase in kidney.31
These effects lead to endothelial dysfunction and animbalance in favor of vasoconstriction. Endo-MR-KO hasno effect on acute CsA–induced nephrotoxicity, indicatingthat Endo-MR is not involved in the deleterious endothelialeffects of CsA, such as a decrease of vascular eNOS
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Figure 3 |Mineralocorticoid receptor-knockout (MR-KO) in smooth muscle cell (SMC) and not in the endothelium prevents thecyclosporine A (CsA)–induced renal overexpression of neutrophil gelatinase–associated lipocalin (NGAL). Renal NGAL expression isinduced by CsA in (a) Endo-MR-KO (endothelial cell-MR-KO) mice but not prevented in (c) SMC-MR-KO mice. Bar = 50 μm. CsA in (b) Endo-MR-KObut not in (d) SMC-MR-KO mice increases renal NGAL protein. Data are expressed as mean± s.e.m. (n=6–9). Two-way analysis of variance(ANOVA) test: **Po0.01 and ***Po0.001 CsA versus VH. Ctl, control; VH, vehicle.
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bas i c resea rch CA Amador et al.: Vascular MR in renal hemodynamics after CsA administration
phosphorylation, a surrogate indicator of eNOS activation.MR deletion in SMC did not affect the CsA–induced decreaseof vascular eNOS phosphorylation, suggesting that thebeneficial effect of MR deletion in vascular SMC isindependent of eNOS activity. This is consistent with studiesin aortic rings treated with CsA, where endothelium-independent relaxation (induced by sodium nitroprusside)was greater than endothelium-dependent relaxation (inducedby acetylcholine),32 suggesting that the overall vascular impactof CsA may be higher in SMC compared with that in theendothelium.
A key finding of the present study using mice specificallydeficient in MR in the vasculature is therefore that SMC-MRdeletion is sufficient to protect the kidney from CIN despite
intact kidney MR function. This suggests that the vasocon-striction is a critical component of the acute toxicity of CsAon the kidney. We do not exclude that CsA also has adeleterious impact on other cell types such as proximaltubular cells, which are not affected by SMC-MR. However,our study supports the idea that renal ischemia caused byCsA–induced vasoconstriction can sensitize the proximaltubule to cell damage induced by CsA. Indeed, we noticedthat most of the tubular vacuolization were observed in thepars recta, a region known to be highly sensitive to hypoxia.33
This also correlates with the beneficial effect of L-type Ca2+
channels blockers in experimental24 and clinical acute CIN,4
an effect that is likely due to inhibition of the vascular sideeffects of CIN and not direct effects on the proximal tubule,
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Figure 4 |Mineralocorticoid receptor-knockout (MR-KO) in smooth muscle cell (SMC) prevents cyclosporine A (CsA)–inducedphosphorylation of contractile proteins. (a) The decrease in endothelial nitric oxide synthase (eNOS) phosphorylation by CsA is similar in Ctland SMC-MR-KO mice. The increase in (b) myosin light-chain kinase (MLCK) and (c) myosin regulatory light chain 2 (MLC2) phosphorylation byCsA is prevented by the ablation of MR in SMC. Data are expressed as mean ± s.e.m. (n= 6–9). Two-way analysis of variance (ANOVA) test.*Po0.05 for the indicated group comparisons. Ctl, control; VH, vehicle.
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CA Amador et al.: Vascular MR in renal hemodynamics after CsA administration bas i c resea rch
where L-type Ca2+ channels have not been reported to beexpressed.
Previous reports showing the beneficial effects of pharma-cological MRAs in experimental models of CIN9–11 couldtherefore be explained by mitigation of the increasedvasoconstrictive response to various vasoactive agents, whichare known to be released locally in the renal vasculature aftercalcineurin inhibition resulting in deleterious renal hemody-namics. Rossi et al.25 showed that part of renal vasoconstric-tion induced by CsA is related to norepinephrine releasedfrom nerve terminals in kidney preparations.25 Indeed, theadministration of the α-adrenergic antagonist phenoxybenza-mine or renal denervation improved RBF and RVR impair-ment caused by CsA treatment in conscious rats.34 Thisvasoconstrictor response elicited by norepinephrine is depen-dent on L-type Ca2+ channel activity, which could thereforebe modulated by SMC-MR.
Owing to difference in CsA pharmacology in humans androdents, rodent preclinical models require higher doses of
CsA to achieve similar pathology, and these doses are notrelevant to human CsA toxicity. Thus, care must be takenwhen extrapolating from rodent models to human disease.Nevertheless, the impact of CsA on renal hemodynamics inhuman kidney allograft recipients and the potential benefit ofcotreatment with L-type Ca2+ channel antagonists has beenhighlighted recently by clinical studies. Indeed, daily CsAadministration in transplant patients is followed by a transientdecrease of the RBF,4 showing a rapid effect of CsA on renalhemodynamics, which in turn can drive the acute nephro-toxicity of transplanted patients treated with CsA. It isimportant to note that the decrease of RBF observed 2 h afterCsA treatment each day is attenuated by cotreatment withCa2+ channel blockers.4 Nankivell et al.35 also demonstratedthat CsA decreased RBF (while tacrolimus, another calcineur-in inhibitor, did not), an effect mitigated by Ca2+ channelblockade. This finding was also reported in non-hypertensiverenal transplant recipients.36 Ca2+ channel blockers werebetter than both ARB37 and ACE-I38 in improving renal
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Figure 5 |Mineralocorticoid receptor-knockout (MR-KO) in smooth muscle cell (SMC) modulates renal vascular resistance through theactivity of L-type Ca2+ channel. Maximum variation in (a and b) mean arterial blood pressure (MAP) and (c and d) renal vascular resistance(RVR) produced by in vivo administration of angiotensin II (AngII) in control (Ctl) and SMC-MR-KO mice treated with cyclosporine A (CsA)(n= 6–9). Contractile response (expressed as relative perfusion pressure) of ex vivo isolated perfused kidney to (e) potassium chloride (KCl) and(f) BayK8644 in Ctl and SMC-MR-KO mice treated with CsA. Vasoconstriction dependent on the L-type Ca2+ channel is blunted in CsA–treatedSMC-MR-KO. Data are expressed as mean± s.e.m. (n= 8). Two-way analysis of variance (ANOVA) test: *Po0.05 vs. Ctl+CsA.
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hemodynamics and glomerular filtration rate, independentlyof beneficial effect on blood pressure control. Ca2+ channelblockers also increased glomerular filtration rate after short-39
and long-40 term use in kidney transplant patients. Theseclinical data were analyzed in a meta-analysis that showedCa2+ channel blockers to be the only vasoactive drugs withproven renal benefit during kidney transplantation.41
Interestingly, improved glomerular filtration rate during Ca2+
channel blocker administration was also reported in CsA–treatedpatients with cardiac transplantation42 and cardiac/lungtransplantation.43 Ca2+ channel blockers also demonstratedrenal benefits in autoimmune diseases such as psoriasis.44
Our results show that SMC-MR activation has a crucialrole in the pathogenesis of acute CIN by modulatingvasoconstriction and increased RVR induced by CsAtreatment. These preclinical data strengthen the rationalefor the mechanistic roles of both the MR and the L-type Ca2+
channel and their interaction in acute CsA nephrotoxicity.
MATERIALS AND METHODSAnimalsGenetic ablation of MR in SMC was obtained as describedpreviously,14 whereas deletion in the endothelium was obtained bymating transgenic mice expressing Cre recombinase in endothelialcells (Tie2-Cre mice) with mice harboring MR–floxed alleles,45
resulting in Endo-MR-KO mice. Littermate Cre-negative mice wereused as controls. Efficient MR ablation was confirmed in whole aortafrom Endo-MR-KO and SMC-MR-KO mice, as compared with Ctlmice, with a reduction of MR mRNA expression of 75% and 50%,respectively (mRNA relative expression; Ctl 1.00± 0.22 vs. Endo-MR-KO 0.25± 0.11; Ctl 1.00± 0.08 vs. SMC-MR-KO 0.55± 07,n= 4–5, Mann–Whitney U-test: *Po0.05 Ctl vs. MR-KO). Thesereductions in the aortic mRNA abundance for MR are similar tothose previously showed by Schäfer et al.46 in Endo-MR-KO mice,and by Galmiche et al.14 in SMC-MR-KO mice. MR protein expressionwas virtually eliminated in SMC from SMC-MR-KO aortas whenendothelium is removed, (Supplementary Figure S3 online). All animalbreeding, housing, and protocols were performed in accordance withthe ethical guidelines of INSERM (Institut National de la Santé et de laRecherche Médicale) for the care and use of laboratory animals. Ourlocal ethical committee for animal experimentations at Charles DarwinUniversity approved all experiments under the Ce5/2012/080 record.All animal experimentations adhered to the NIH Guide for Care andUse for the Laboratory Animals.
Experimental protocolEight-week-old C57BL/6 transgenic female mice, with conditionalinactivation of MR in either Endo (Endo-MR-KO) or SMC(SMC-MR-KO), were fed 7 days with low-salt diet (0.01% NaCl)to sensitize the renin–angiotensin–aldosterone system, as describedpreviously.9 After 1 week, mice were treated with vehicle (EtOH 95%and Cremophor Sigma, in a relation 1:4 (v/v)) or CsA (100 mg/kgper day diluted in vehicle solution) subcutaneously during 2 days.This dose of CsA was lower in comparison with the study of Siedleckiet al.,47 but sufficient to induce alterations in renal function andrenal structure that mimics the pathological lesions in human acuteCIN.17 Of note, the dose of CsA required to induce nephrotoxicity inpreclinical rodent models is higher compared with those classicallyused in human for therapeutic objectives.
Biochemical assaysAt killing, blood was collected in specific tubes and centrifuged toobtain plasma, and then stored at − 20 °C. Plasma urea andcreatinine were analyzed by an enzymatic method using a Konelabv.7.0.1 automate (Pierce/Thermo Fischer Scientific, Rockford, IL).
Western blottingAbdominal aortas and kidneys were collected and proteins freshlyextracted with a sodium dodecyl sulfate 1% buffer pH= 7.4.Proteins were separated by sodium dodecyl sulfate-polyacrylamidegel electrophoresis (Bio-Rad, Hercules, CA), blotted ontonitrocellulose membranes (Amersham ECL Plus; GE HealthcareLife Sciences, Freiburg, Germany), and probed with primaryantibodies: anti-NGAL, anti-peNOS, β-actin (Abcam, Cambridge,UK), anti-eNOS (Santa Cruz Biotechnology, Santa Cruz, CA), anti-pMLCK (Life Technologies Corporation, Carlsbad, CA),anti-MLCK (Sigma-Aldrich, St Louis, MO), anti-pMLC2, anti-MLC2 (Cell Signaling, Boston, MA), and anti-MR (gift of Prof.Celso Gomez-Sanchez, University of Mississippi Medical Center,Jackson, MS). Secondary antibody anti-rabbit HRP or anti-mouseHRP (GE Healthcare Life Sciences) was used. Specific binding wasdetected using enhanced chemiluminescence (Amersham ECL Plus;GE Healthcare Life Sciences) and exposed in a Fujifilm Lumines-cent Image Analyzer LAS4000 System (Tokyo, Japan). Images ofblots were quantified by densitometry analysis (ImageJ 1.43u, USNational Institutes of Health, Bethesda, MD).
Real-time PCRThoracic aortas were collected and total RNA was extracted withTRIzol Reagent, and cDNA was produced from RNA usingSuperscript II Reverse Transcriptase Kit (all from Life TechnologiesCorporation, Carlsbad, CA). Real-time PCR reactions wereperformed using a Bio-Rad Thermal Cycler (Cergy-St-Christophe,France) (iCycler iQ apparatus) and transcript levels were detected bySYBR Green method. The sequences of the mouse primer pairs arethe following: 18S, (F) 5′-CGCCGCTAGAGGTGAAATTC-3′, (R)5′-TCTTGGCAAATGCTTTCGC-3′; MR, (F) 5′-CCAGAAGAGGGGACCACATA-3′, (R) 5′-GGAATTGTCGTAGCCTGCAT-3′; Cav1.2,(F) 5′-ATGAAAACACGAGGATGTACGTT-3′, (R) 5′-ACTGACGGTAGAGATGGTTGC-3′. All PCR products were subjected tomelting-curve program to confirm amplification specificity. Resultswere analyzed according to the standard curve method, and mRNAabundance was calculated to the amount of 18S for each sample.
Histological analysis and immunohistochemistryKidneys were fixed in Bouin solution (Reactifs RAL, Martillac,France) or paraformaldehyde 4% overnight before being includedinto paraffin blocks. Four-micrometer sections were performed forhematoxylin–eosin staining (Bouin fixation) and for immunohis-tochemistry of NGAL. Tubular vacuolization was scored with asemiquantitative score by two pathologists blinded to the experi-mental groups. Kidney sections were incubated with anti-NGALantibody (R&D Systems, Minneapolis, MN) before anti-immunoglobulin G secondary antibody for DAB (3,3'-diaminoben-zidine) peroxidase revelation.
Renal hemodynamicsAfter CsA treatment, mice were anesthetized by pentobarbitalsodium (50–60 mg/kg body weight intraperitoneally; Nembutal;Abbott, Chicago, IL) and moved to a servo-controlled table kept at
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CA Amador et al.: Vascular MR in renal hemodynamics after CsA administration bas i c resea rch
37 °C. The left femoral artery was catheterized for measurement ofarterial pressure, and a femoral venous catheter was used for infusionof volume replacement. Bovine serum albumin (4.75 g/dl of salinesolution) was infused initially at 50 μl/min to replace surgical losses,and then at 10 μl/min for maintenance. Arterial pressure wasmeasured via a pressure transducer in left femoral artery(Statham P23 DB, Gould, Valley View, OH), and renal blood flowwas measured by a flowmeter (0.5v probe; Transonic systems TS420,Ithaca, NY). In vivo RVR was obtained from the relationship betweenblood pressure and renal blood flow, after intravenous injections ofAngII (0.5, 1, and 2 ng; Sigma-Aldrich).
Isolated perfused kidneyAfter CsA treatment, the left kidney was isolated and renal artery wascannulated and perfused at 1 ml/min with a Krebs–Henseleitsolution (mmol/l: 118.4 NaCl, 4.7 KCl, 2 CaCl2, 1.2 MgSO4,1.2 KH2PO4, 20 NaHCO3, and 11 glucose). The perfusion buffer wasmaintained at 37 °C and oxygenated (95% O2/5% CO2). After a 30-min equilibration period, the vasoactive response of the renalmicrovasculature to BayK8644 (0.1 μmol/l; Sigma-Aldrich) and toincreasing concentrations of KCl (0–90 mmol/l) was tested. Changesin perfusion pressures of the renal artery ex vivo were measured witha pressure transducer linked to a PowerLab digital data recorder(PowerLab Chart, ADInstrument-Europe, UK).
StatisticsAll data are expressed as the mean± s.e.m. Data were analyzed bytwo-way analysis of variance followed by Dunn post hoc test(42 groups). Mann–Whitney nonparametric test (2 groups), orlineal regression and comparison between slopes for renal hemody-namics. All analyses were performed using GraphPad Prism V6.01(GraphPad Software, San Diego, CA). P-values o0.05 wereconsidered as statistically significant.
DISCLOSUREAll the authors declared no competing interests.
ACKNOWLEDGMENTSWe thank Nicolette Farman for her meaningful suggestions. This workwas supported by INSERM and grants from the ‘Agence Nationalepour la Recherche’ (ANR09-BLAN-0156-01), the ‘Centre de RechercheIndustrielle et Technique’ and the FP7-funded COST-ADMIRE BM1301network. FJ received a fellowship from the Philippe Foundationduring his sabbatical in IZJ laboratory. CAA was supported by‘CONICYT, Beca de Postdoctorado en el Extranjero, Becas-Chile’ No.74130051. GA-G was supported by a CORRDIM-Ile de Francefellowship.
SUPPLEMENTARY MATERIALFigure S1. MR-KO in endothelium does not prevent CsA–inducedactivation of contractile proteins.Figure S2. CsA does not modify Cav1.2 mRNA abundance both aortaand whole kidney.Figure S3. Western blot for MR in aortas of control (Ctl) and SMC-MR-KO mice without endothelium (removed by rubbing the intima withpincers, upper panel).Supplementary material is linked to the online version of the paper athttp://www.nature.com/ki
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