HHS Public Access Author manuscript Author Manuscript
Kidney Int. Author manuscript; available in PMC 2017 February 01. Published in final edited form as: Kidney Int. 2016 February ; 89(2): 354–362. doi:10.1038/ki.2015.312.
Deletion of mineralocorticoid receptors in smooth muscle cells blunts renal vascular resistance following acute cyclosporine administration
Author Manuscript
Cristian 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, Reims University Hospital, Reims, France
3Hemodynamic 4Pathology 5German
Platform, Tenon Hospital, Paris, France
Department, Necker Hospital, Paris, France
Cancer Research Center, Heidelberg, Germany
6University
of Alabama at Birmingham, Birmingham, Alabama, USA
7Molecular
Cardiology Research Institute, Tufts Medical Center, Boston, Massachusetts, USA
Abstract Author Manuscript Author Manuscript
Calcineurin inhibitors such as cyclosporine A (CsA) are still commonly used after renal transplantation, despite CsA–induced nephrotoxicity (CIN), which is partly related to vasoactive mechanisms. The mineralocorticoid receptor (MR) is now recognized as a key player in the control of vascular tone, and both endothelial cell- and vascular smooth muscle cell (SMC)-MR modulate the vasoactive responses to vasodilators and vasoconstrictors. Here we tested whether vascular MR is involved in renal hemodynamic changes induced by CsA. The relative contribution of vascular MR in acute CsA treatment was evaluated using mouse models with targeted deletion of MR in endothelial cell or SMC. Results indicate that MR expressed in SMC, but not in endothelium, contributes to the increase of plasma urea and creatinine, the appearance of isometric tubular vacuolization, and overexpression of a kidney injury biomarker (neutrophil gelatinase– associated lipocalin) after CsA treatment. Inactivation of MR in SMC blunted CsA–induced phosphorylation of contractile proteins. Finally, the in vivo increase of renal vascular resistance induced by CsA was blunted when MR was deleted from SMC cells, and this was associated with decreased L-type Ca2+ channel activity. Thus, our study provides new insights into the role of
Correspondence: Frederic Jaisser, INSERM UMRS 1138, Team 1, Research Centre of Cordeliers, 15 rue de l’école de médecine, Paris 75006, France. [email protected]. 8These first two authors contributed equally to this work. DISCLOSURE All the authors declared no competing interests. SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at www.kidney-international.org.
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vascular MR in renal hemodynamics during acute CIN, and provides rationale for clinical studies of MR antagonism to manage the side effects of calcineurin inhibitors.
Keywords acute kidney injury; aldosterone; calcineurin inhibitors; nephrotoxicity
Author Manuscript
Calcineurin inhibitors, such as cyclosporine A (CsA) and tacrolimus, are immunosuppressive drugs widely used after renal transplantation. However, calcineurin inhibitors induce acute nephrotoxicity that can result in kidney failure.1 Although the mechanisms underlying CsA–induced nephrotoxicity (CIN) remain unclear,2 alteration of renal hemodynamics appears central in acute CIN, and renal vasoconstriction has been reported as an initial event linked to CIN. CsA promotes renal afferent arteriolar vasoconstriction in rats, a pathogenic factor at least as important as tubular injury in acute CsA nephrotoxicity.3 In kidney allograft recipients, daily CsA administration is followed by a transient decrease of the renal blood flow (RBF),4 suggesting a rapid effect of CsA on renal hemodynamics. These effects are sensitive to angiotensin-converting enzyme inhibitors5 or L-type Ca2+ channel antagonists.4 CsA increases noradrenaline-, angiotensin II (AngII)–, endothelin-, and vasopressin-induced vasoconstriction in rat arteries.6–8 Despite these limitations, CsA is still very commonly used after organ transplantation, especially in low-income countries. Therefore, deciphering the mechanisms by which CsA promotes renal hemodynamic alterations is crucial to the development of new therapeutic strategies to prevent or ameliorate acute CIN.
Author Manuscript
Pharmacological blockade of mineralocorticoid receptor (MR) is highly effective in experimental models of CIN.9–11 The beneficial effects of MR antagonism include regulation of renal remodeling (apoptosis, fibrosis) and modulation of vasoactive factors, which are increased during CsA treatment.12 However, the cellular targets of the MR antagonists (MRAs) and the underlying mechanisms related to the vasoconstriction observed in acute CIN are still unknown. Recent studies have shown that the MR has a central role in the control of vascular tone: both endothelial cell (Endo)- and vascular smooth muscle cell (SMC)-MR modulates the vasoactive responses to vasodilators and vasoconstrictors.13–16 The aim of this study was therefore to analyze whether the vascular MR is involved in the renal hemodynamic changes induced by CsA, and if so, to evaluate the relative contribution of the Endo- or vascular SMC-MR in this effect.
RESULTS Author Manuscript
MR gene inactivation in SMC but not in Endo prevents acute kidney failure, tubular vacuolization, and NGAL expression induced by acute CsA To determine whether vascular Endo- or SMC-MR is involved in 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 progressive body weight loss in Ctl, Endo-MR-KO, and SMC-MR-KO mice (Figure 1a and b). Plasma urea and creatinine were increased after 2 days of CsA treatment in Ctl mice (Figure 1c–f) and in Endo-MR-KO
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mice to a similar extent (Figure 1c and e). However, specific deletion of MR in SMC prevented this increase (Ctl+CsA vs. SMC-MR-KO+CsA: uremia 21.2 ± 5 vs. 8.1 ± 0.8 mmol/l, creatininemia 33.7 ± 9.5 vs. 10.4 ± 0.6 mmol/l; P < 0.05; Figure 1d and f).
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CsA–treated Ctl mice developed isometric vacuolization of the proximal tubule (Figure 2a– d), similar to the pathology previously described in post-transplantation patients with acute CIN17 and in other CIN experimental models.11,18,19 MR deletion in SMC (Figure 2c and d) prevented the development of these histological lesions, whereas MR deletion in Endo had no effect (Figure 2a and b). Additionally, immunohistochemistry in renal proximal tubules of Ctl and Endo-MR-KO demonstrated strong expression of neutrophil gelatinase–associated lipocalin (NGAL, or Lcn2 in mice), a small glycoprotein used as marker of tubular injury in mice and humans,20 after 2 days of CsA treatment (Figure 3a and c). NGAL protein expression was induced in Ctl and Endo-MR-KO as determined by western blotting of whole kidneys (Figure 3b). MR-KO in SMC prevented the NGAL overexpression induced by CsA (Figure 3d). MR-KO in SMC prevents CsA–induced phosphorylation of vascular smooth muscle contractile proteins and modulates renal vascular resistance through the activity of L-type Ca2+ channel
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As the MR expressed in SMC, but not in endothelial cells, was critical for acute kidney injury after CsA administration, and as the vasoconstriction has been proposed as a critical mechanism for acute CIN,2,3,4,11 we explored whether (i) CsA affected 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 of eNOS at Ser1177, is decreased after CsA treatment to a similar extent 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 SMC contraction.21 The phosphorylation levels of MLCK as well as MLC2 proteins were increased in abdominal aortas of CsA–treated Ctl mice (Figure 4b and c) and Endo-MR-KO mice (Supplementary Figure S1a and 1b online). However, MR deletion in SMC prevented the effect of CsA on both MLCK and MLC2 phosphorylation (Figure 4b and c), thus altering a key mechanism in CsA–induced SMC contraction.
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Previous clinical observations in transplanted patients indicated that renal vascular resistance (RVR) is increased in patients shortly after oral CsA administration.4,22 To establish a direct role of SMC-MR in the CsA effects on the intrarenal vasculature, we studied changes of blood pressure and RVR in CsA–treated mice challenged with AngII to induce a vasoconstrictive response in vivo. Baseline mean arterial blood pressure was similar in Ctl and SMC-MR-KO mice treated with CsA (Ctl+CsA: 81 ± 3 mmHg; SMC-MR-KO+CsA: 79 ± 3 mmHg) (Figure 5a). AngII similarly increases mean arterial blood pressure in Ctl and SMC-MR-KO mice (Figure 5b). In vivo administration of Angll increased RVR in a dosedependent manner in Ctl mice (Figure 5d). However, the increase in RVR induced by Angll is blunted in SMC-MR-KO mice compared with Ctl mice (Figure 5c and d).
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L-type Ca2+ channels (Cav1.2) are direct targets of MR in the SMC:15 SMC-MR deletion blunted the activation of L-type Ca2+ channel in mesenteric arteries.15 As CsA was previously shown to increase Ca2+ signaling in the vasculature by increasing the activity of the membrane Cav1.2 channel,23 we hypothesized that the modulation of Cav1.2 activity in the renal microvasculature by MR may contribute to the beneficial effects we observed in SMC-MR-KO mice treated by CsA. We therefore analyzed the effects of KCl (which allows depolarization-induced L-type Ca2+ channel activation) and of BayK8644 (a specific L-type Ca2+ channel agonist) on the renal microvasculature perfusion pressure in isolated kidneys after CsA treatment. Renal arteries from SMC-MR-KO kidneys showed an attenuated contractile response to KCl (Figure 5e) and to BayK8644 (Figure 5f) compared with Ctl kidneys. This demonstrated that in SMC-MR-KO mice, the renal vascular activity of the Ltype Ca2+ channel is blunted after CsA treatment.
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DISCUSSION Our data indicate that the SMC-MR, but not the Endo-MR, is mandatory for acute kidney failure, tubular vacuolization, and NGAL overexpression induced by CsA administration. These effects are associated to a decreased vasoconstrictive response in CsA–treated SMCMR-KO mice that is associated with blunted vascular L-type Ca2+ channel activity.
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The alteration in renal hemodynamics has a pivotal role in the acute nephrotoxicity of transplanted patients treated with CsA as renal vasoconstriction has been established as an initial event linked to acute CIN.4,5,22 In preclinical models, CsA promotes renal afferent arteriolar vasoconstriction3 and increases the vasoactive response to several vasoconstrictors.6–8 Additionally, CsA induces a Ca2+ influx–dependent vasoconstriction in isolated mesenteric arteries,23 and increases the perfusion pressure of the caudal artery.24 This arterial vasoconstriction depends on extracellular Ca2+.23 It has been proposed that CsA enhances signal transmission between G-protein-coupled receptors and L-type Ca2+ channel.24 CsA induces Ca2+ influx in vascular SMC through the IP3 pathway by modulating the phosphorylation of IP3 receptor.6 Interestingly, the use of Ca2+ channel blockers blunts these effects induced by CsA.23–25 In the present study, we showed in CsA– treated mice that SMC-MR deletion prevents: (1) renal microvascular contraction, (2) phosphorylation of contractile proteins in SMC, and (3) vascular L-type Ca2+ channel activity. Both CsA and the MR expressed in vascular SMC affect the Ca2+ signaling pathway, which is essential for SMC contractility. McCurley et al.15 recently demonstrated that vascular SMC-MR modulates the expression of the L-type Ca2+ channel Cav1.2 and that SMC-MR deletion blunts the vasoconstrictive effect of a Cav1.2 agonist. In cardiomyocytes, another contractile cell type where MR has an essential role, MR modulates the activity of both Cav1.2 and the ryanodine receptor, affecting cardiomyocyte contraction.26,27 In the present study, we showed that the effect of SMC-MR deletion on Ltype Ca2+ activity is independent of the abundance of Cav1.2 expression (Supplementary Figure S2a and b online). Furthermore, by modulating cellular Ca2+ homeostasis, SMC-MR will affect downstream signaling to the contractile machinery. Therefore, we propose that vascular SMC-MR, by modulating L-type Ca2+ channel activity and the phosphorylation of proteins critical for SMC contraction, is a key regulator of microvascular contraction and renal hemodynamics after CsA administration. Kidney Int. Author manuscript; available in PMC 2017 February 01.
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CsA administration has been reported to decrease vascular NO production,28 by increasing the synthesis of superoxide anion in renal endothelial cells,29 and decreasing the activity of eNOS,30 catalase, and glutathione peroxidase in kidney.31 These effects lead to endothelial dysfunction and an imbalance in favor of vasoconstriction. Endo-MR-KO has no effect on acute CsA–induced nephrotoxicity, indicating that Endo-MR is not involved in the deleterious endothelial effects of CsA, such as a decrease of vascular eNOS phosphorylation, a surrogate indicator of eNOS activation. MR deletion in SMC did not affect the CsA–induced decrease of vascular eNOS phosphorylation, suggesting that the beneficial effect of MR deletion in vascular SMC is independent of eNOS activity. This is consistent with studies in aortic rings treated with CsA, where endothelium-independent relaxation (induced by sodium nitroprusside) was greater than endothelium-dependent relaxation (induced by acetylcholine),32 suggesting that the overall vascular impact of CsA may be higher in SMC compared with that in the endothelium.
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A key finding of the present study using mice specifically deficient in MR in the vasculature is therefore that SMC-MR deletion is sufficient to protect the kidney from CIN despite intact kidney MR function. This suggests that the vasoconstriction is a critical component of the acute toxicity of CsA on the kidney. We do not exclude that CsA also has a deleterious impact on other cell types such as proximal tubular cells, which are not affected by SMCMR. However, our study supports the idea that renal ischemia caused by CsA–induced vasoconstriction can sensitize the proximal tubule to cell damage induced by CsA. Indeed, we noticed that most of the tubular vacuolization were observed in the pars 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 side effects of CIN and not direct effects on the proximal tubule, where L-type Ca2+ channels have not been reported to be expressed. Previous reports showing the beneficial effects of pharmacological MRAs in experimental models of CIN9–11 could therefore be explained by mitigation of the increased vasoconstrictive response to various vasoactive agents, which are known to be released locally in the renal vasculature after calcineurin inhibition resulting in deleterious renal hemodynamics. Rossi et al.25 showed that part of renal vasoconstriction induced by CsA is related to norepinephrine released from nerve terminals in kidney preparations.25 Indeed, the administration of the a-adrenergic antagonist phenoxybenzamine or renal denervation improved RBF and RVR impairment caused by CsA treatment in conscious rats.34 This vasoconstrictor response elicited by norepinephrine is dependent on L-type Ca2+ channel activity, which could therefore be modulated by SMC-MR.
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Owing to difference in CsA pharmacology in humans and rodents, rodent preclinical models require higher doses of CsA to achieve similar pathology, and these doses are not relevant to human CsA toxicity. Thus, care must be taken when extrapolating from rodent models to human disease. Nevertheless, the impact of CsA on renal hemodynamics in human kidney allograft recipients and the potential benefit of cotreatment with L-type Ca2+ channel antagonists has been highlighted recently by clinical studies. Indeed, daily CsA administration in transplant patients is followed by a transient decrease of the RBF,4 showing a rapid effect of CsA on renal hemodynamics, which in turn can drive the acute
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nephrotoxicity of transplanted patients treated with CsA. It is important to note that the decrease of RBF observed 2 h after CsA treatment each day is attenuated by cotreatment with Ca2+ channel blockers.4 Nankivell et al.35 also demonstrated that CsA decreased RBF (while tacrolimus, another calcineurin inhibitor, did not), an effect mitigated by Ca2+ channel blockade. This finding was also reported in non-hypertensive renal transplant recipients.36 Ca2+ channel blockers were better than both ARB37 and ACE-I38 in improving renal hemodynamics and glomerular filtration rate, independently of beneficial effect on blood pressure control. Ca2+ channel blockers also increased glomerular filtration rate after short-39 and long-40 term use in kidney transplant patients. These clinical data were analyzed in a meta-analysis that showed Ca2+ channel blockers to be the only vasoactive drugs with proven renal benefit during kidney transplantation.41 Interestingly, improved glomerular filtration rate during Ca2+ channel blocker administration was also reported in CsA–treated patients with cardiac transplantation42 and cardiac/lung transplantation.43 Ca2+ channel blockers also demonstrated renal benefits in autoimmune diseases such as psoriasis.44
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Our results show that SMC-MR activation has a crucial role in the pathogenesis of acute CIN by modulating vasoconstriction and increased RVR induced by CsA treatment. These preclinical data strengthen the rationale for the mechanistic roles of both the MR and the Ltype Ca2+ channel and their interaction in acute CsA nephrotoxicity.
MATERIALS AND METHODS Animals
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Genetic ablation of MR in SMC was obtained as described previously,14 whereas deletion in the endothelium was obtained by mating transgenic mice expressing Cre recombinase in endothelial cells (Tie2-Cre mice) with mice harboring MR–floxed alleles,45 resulting in Endo-MR-KO mice. Littermate Cre-negative mice were used as controls. Efficient MR ablation was confirmed in whole aorta from Endo-MR-KO and SMC-MR-KO mice, as compared with Ctl mice, 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: *P < 0.05 Ctl vs. MR-KO). These reductions in the aortic mRNA abundance for MR are similar to those 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 expression was virtually eliminated in SMC from SMCMR-KO aortas when endothelium is removed, (Supplementary Figure S3 online). All animal breeding, housing, and protocols were performed in accordance with the ethical guidelines of INSERM (Institut National de la Santé et de la Recherche Médicale) for the care and use of laboratory animals. Our local ethical committee for animal experimentations at Charles Darwin University approved all experiments under the Ce5/2012/080 record. All animal experimentations adhered to the NIH Guide for Care and Use for the Laboratory Animals. Experimental protocol Eight-week-old C57BL/6 transgenic female mice, with conditional inactivation 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 described
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previously.9 After 1 week, mice were treated with vehicle (EtOH 95% and Cremophor Sigma, in a relation 1:4 (v/v)) or CsA (100mg/kg per day diluted in vehicle solution) subcutaneously during 2 days. This dose of CsA was lower in comparison with the study of Siedlecki et al.,47 but sufficient to induce alterations in renal function and renal structure that mimics the pathological lesions in human acute CIN.17 Of note, the dose of CsA required to induce nephrotoxicity in preclinical rodent models is higher compared with those classically used in human for therapeutic objectives. Biochemical assays At killing, blood was collected in specific tubes and centrifuged to obtain plasma, and then stored at −20 °C. Plasma urea and creatinine were analyzed by an enzymatic method using a Konelab v.7.0.1 automate (Pierce/Thermo Fischer Scientific, Rockford, IL).
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Western blotting
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Abdominal aortas and kidneys were collected and proteins freshly extracted with a sodium dodecyl sulfate 1% buffer pH = 7.4. Proteins were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA), blotted onto nitrocellulose membranes (Amersham ECL Plus; GE Healthcare Life Sciences, Freiburg, Germany), and probed with primary antibodies: anti-NGAL, anti-peNOS, ß-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), antipMLC2, anti-MLC2 (Cell Signaling, Boston, MA), and anti-MR (gift of Prof. Celso GomezSanchez, University of Mississippi Medical Center, Jackson, MS). Secondary antibody antirabbit HRP or anti-mouse HRP (GE Healthcare Life Sciences) was used. Specific binding was detected using enhanced chemiluminescence (Amersham ECL Plus; GE Healthcare Life Sciences) and exposed in a Fujifilm Luminescent Image Analyzer LAS4000 System (Tokyo, Japan). Images of blots were quantified by densitometry analysis (ImageJ 1.43u, US National Institutes of Health, Bethesda, MD). Real-time PCR
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Thoracic aortas were collected and total RNA was extracted with TRIzol Reagent, and cDNA was produced from RNA using Superscript II Reverse Transcriptase Kit (all from Life Technologies Corporation, Carlsbad, CA). Real-time PCR reactions were performed using a Bio-Rad Thermal Cycler (Cergy-St-Christophe, France) (iCycler iQ apparatus) and transcript levels were detected by SYBR Green method. The sequences of the mouse primer pairs are the following: 18S, (F) 5′-CGCCGCTAGAGGTGAAATTC-3′, (R) 5′TCTTGGCAAATGCTTTCGC-3′; MR, (F) 5′-CCAGAAGAGGG GACCACATA-3′, (R) 5′-GGAATTGTCGTAGCCTGCAT-3′; Cav1.2, (F) 5′ATGAAAACACGAGGATGTACGTT-3′, (R) 5′-ACTGACGGTAGAGATGGTTGC-3′. All PCR products were subjected to melting-curve program to confirm amplification specificity. Results were analyzed according to the standard curve method, and mRNA abundance was calculated to the amount of 18S for each sample.
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Histological analysis and immunohistochemistry
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Kidneys were fixed in Bouin solution (Reactifs RAL, Martillac, France) or paraformaldehyde 4% overnight before being included into paraffin blocks. Fourmicrometer sections were performed for hematoxylin–eosin staining (Bouin fixation) and for immunohistochemistry of NGAL. Tubular vacuolization was scored with a semiquantitative score by two pathologists blinded to the experimental groups. Kidney sections were incubated with anti-NGAL antibody (R&D Systems, Minneapolis, MN) before antiimmunoglobulin G secondary antibody for DAB (3,3′-diaminobenzidine) peroxidase revelation. Renal hemodynamics
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After CsA treatment, mice were anesthetized by pentobarbital sodium (50–60 mg/kg body weight intraperitoneally; Nembutal; Abbott, Chicago, IL) and moved to a servo-controlled table kept at 37 °C. The left femoral artery was catheterized for measurement of arterial pressure, and a femoral venous catheter was used for infusion of volume replacement. Bovine serum albumin (4.75 g/dl of saline solution) was infused initially at 50 μl/min to replace surgical losses, and then at 10 μl/min for maintenance. Arterial pressure was measured via a pressure transducer in left femoral artery (Statham P23 DB, Gould, Valley View, OH), and renal blood flow was measured by a flowmeter (0.5v probe; Transonic systems TS420, Ithaca, NY). In vivo RVR was obtained from the relationship between blood pressure and renal blood flow, after intravenous injections of AngII (0.5, 1, and 2 ng; SigmaAldrich). Isolated perfused kidney
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After CsA treatment, the left kidney was isolated and renal artery was cannulated and perfused at 1 ml/min with a Krebs–Henseleit solution (mmol/l: 118.4 NaCl, 4.7 KCl, 2 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 20 NaHCO3, and 11 glucose). The perfusion buffer was maintained at 37 °C and oxygenated (95% O2/5% CO2). After a 30-min equilibration period, the vasoactive response of the renal microvasculature to BayK8644 (0.1 μmol/l; SigmaAldrich) and to increasing concentrations of KCl (0–90 mmol/l) was tested. Changes in perfusion pressures of the renal artery ex vivo were measured with a pressure transducer linked to a PowerLab digital data recorder (PowerLab Chart, ADInstrument-Europe, UK). Statistics
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All data are expressed as the mean ± s.e.m. Data were analyzed by two-way analysis of variance followed by Dunn post hoc test (>2 groups). Mann–Whitney nonparametric test (2 groups), or lineal regression and comparison between slopes for renal hemodynamics. All analyses were performed using GraphPad Prism V6.01 (GraphPad Software, San Diego, CA). P-values
Kidney Int. Author manuscript; available in PMC 2017 February 01. Published in final edited form as: Kidney Int. 2016 February ; 89(2): 354–362. doi:10.1038/ki.2015.312.
Deletion of mineralocorticoid receptors in smooth muscle cells blunts renal vascular resistance following acute cyclosporine administration
Author Manuscript
Cristian 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, Reims University Hospital, Reims, France
3Hemodynamic 4Pathology 5German
Platform, Tenon Hospital, Paris, France
Department, Necker Hospital, Paris, France
Cancer Research Center, Heidelberg, Germany
6University
of Alabama at Birmingham, Birmingham, Alabama, USA
7Molecular
Cardiology Research Institute, Tufts Medical Center, Boston, Massachusetts, USA
Abstract Author Manuscript Author Manuscript
Calcineurin inhibitors such as cyclosporine A (CsA) are still commonly used after renal transplantation, despite CsA–induced nephrotoxicity (CIN), which is partly related to vasoactive mechanisms. The mineralocorticoid receptor (MR) is now recognized as a key player in the control of vascular tone, and both endothelial cell- and vascular smooth muscle cell (SMC)-MR modulate the vasoactive responses to vasodilators and vasoconstrictors. Here we tested whether vascular MR is involved in renal hemodynamic changes induced by CsA. The relative contribution of vascular MR in acute CsA treatment was evaluated using mouse models with targeted deletion of MR in endothelial cell or SMC. Results indicate that MR expressed in SMC, but not in endothelium, contributes to the increase of plasma urea and creatinine, the appearance of isometric tubular vacuolization, and overexpression of a kidney injury biomarker (neutrophil gelatinase– associated lipocalin) after CsA treatment. Inactivation of MR in SMC blunted CsA–induced phosphorylation of contractile proteins. Finally, the in vivo increase of renal vascular resistance induced by CsA was blunted when MR was deleted from SMC cells, and this was associated with decreased L-type Ca2+ channel activity. Thus, our study provides new insights into the role of
Correspondence: Frederic Jaisser, INSERM UMRS 1138, Team 1, Research Centre of Cordeliers, 15 rue de l’école de médecine, Paris 75006, France. [email protected]. 8These first two authors contributed equally to this work. DISCLOSURE All the authors declared no competing interests. SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at www.kidney-international.org.
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vascular MR in renal hemodynamics during acute CIN, and provides rationale for clinical studies of MR antagonism to manage the side effects of calcineurin inhibitors.
Keywords acute kidney injury; aldosterone; calcineurin inhibitors; nephrotoxicity
Author Manuscript
Calcineurin inhibitors, such as cyclosporine A (CsA) and tacrolimus, are immunosuppressive drugs widely used after renal transplantation. However, calcineurin inhibitors induce acute nephrotoxicity that can result in kidney failure.1 Although the mechanisms underlying CsA–induced nephrotoxicity (CIN) remain unclear,2 alteration of renal hemodynamics appears central in acute CIN, and renal vasoconstriction has been reported as an initial event linked to CIN. CsA promotes renal afferent arteriolar vasoconstriction in rats, a pathogenic factor at least as important as tubular injury in acute CsA nephrotoxicity.3 In kidney allograft recipients, daily CsA administration is followed by a transient decrease of the renal blood flow (RBF),4 suggesting a rapid effect of CsA on renal hemodynamics. These effects are sensitive to angiotensin-converting enzyme inhibitors5 or L-type Ca2+ channel antagonists.4 CsA increases noradrenaline-, angiotensin II (AngII)–, endothelin-, and vasopressin-induced vasoconstriction in rat arteries.6–8 Despite these limitations, CsA is still very commonly used after organ transplantation, especially in low-income countries. Therefore, deciphering the mechanisms by which CsA promotes renal hemodynamic alterations is crucial to the development of new therapeutic strategies to prevent or ameliorate acute CIN.
Author Manuscript
Pharmacological blockade of mineralocorticoid receptor (MR) is highly effective in experimental models of CIN.9–11 The beneficial effects of MR antagonism include regulation of renal remodeling (apoptosis, fibrosis) and modulation of vasoactive factors, which are increased during CsA treatment.12 However, the cellular targets of the MR antagonists (MRAs) and the underlying mechanisms related to the vasoconstriction observed in acute CIN are still unknown. Recent studies have shown that the MR has a central role in the control of vascular tone: both endothelial cell (Endo)- and vascular smooth muscle cell (SMC)-MR modulates the vasoactive responses to vasodilators and vasoconstrictors.13–16 The aim of this study was therefore to analyze whether the vascular MR is involved in the renal hemodynamic changes induced by CsA, and if so, to evaluate the relative contribution of the Endo- or vascular SMC-MR in this effect.
RESULTS Author Manuscript
MR gene inactivation in SMC but not in Endo prevents acute kidney failure, tubular vacuolization, and NGAL expression induced by acute CsA To determine whether vascular Endo- or SMC-MR is involved in 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 progressive body weight loss in Ctl, Endo-MR-KO, and SMC-MR-KO mice (Figure 1a and b). Plasma urea and creatinine were increased after 2 days of CsA treatment in Ctl mice (Figure 1c–f) and in Endo-MR-KO
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mice to a similar extent (Figure 1c and e). However, specific deletion of MR in SMC prevented this increase (Ctl+CsA vs. SMC-MR-KO+CsA: uremia 21.2 ± 5 vs. 8.1 ± 0.8 mmol/l, creatininemia 33.7 ± 9.5 vs. 10.4 ± 0.6 mmol/l; P < 0.05; Figure 1d and f).
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CsA–treated Ctl mice developed isometric vacuolization of the proximal tubule (Figure 2a– d), similar to the pathology previously described in post-transplantation patients with acute CIN17 and in other CIN experimental models.11,18,19 MR deletion in SMC (Figure 2c and d) prevented the development of these histological lesions, whereas MR deletion in Endo had no effect (Figure 2a and b). Additionally, immunohistochemistry in renal proximal tubules of Ctl and Endo-MR-KO demonstrated strong expression of neutrophil gelatinase–associated lipocalin (NGAL, or Lcn2 in mice), a small glycoprotein used as marker of tubular injury in mice and humans,20 after 2 days of CsA treatment (Figure 3a and c). NGAL protein expression was induced in Ctl and Endo-MR-KO as determined by western blotting of whole kidneys (Figure 3b). MR-KO in SMC prevented the NGAL overexpression induced by CsA (Figure 3d). MR-KO in SMC prevents CsA–induced phosphorylation of vascular smooth muscle contractile proteins and modulates renal vascular resistance through the activity of L-type Ca2+ channel
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As the MR expressed in SMC, but not in endothelial cells, was critical for acute kidney injury after CsA administration, and as the vasoconstriction has been proposed as a critical mechanism for acute CIN,2,3,4,11 we explored whether (i) CsA affected 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 of eNOS at Ser1177, is decreased after CsA treatment to a similar extent 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 SMC contraction.21 The phosphorylation levels of MLCK as well as MLC2 proteins were increased in abdominal aortas of CsA–treated Ctl mice (Figure 4b and c) and Endo-MR-KO mice (Supplementary Figure S1a and 1b online). However, MR deletion in SMC prevented the effect of CsA on both MLCK and MLC2 phosphorylation (Figure 4b and c), thus altering a key mechanism in CsA–induced SMC contraction.
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Previous clinical observations in transplanted patients indicated that renal vascular resistance (RVR) is increased in patients shortly after oral CsA administration.4,22 To establish a direct role of SMC-MR in the CsA effects on the intrarenal vasculature, we studied changes of blood pressure and RVR in CsA–treated mice challenged with AngII to induce a vasoconstrictive response in vivo. Baseline mean arterial blood pressure was similar in Ctl and SMC-MR-KO mice treated with CsA (Ctl+CsA: 81 ± 3 mmHg; SMC-MR-KO+CsA: 79 ± 3 mmHg) (Figure 5a). AngII similarly increases mean arterial blood pressure in Ctl and SMC-MR-KO mice (Figure 5b). In vivo administration of Angll increased RVR in a dosedependent manner in Ctl mice (Figure 5d). However, the increase in RVR induced by Angll is blunted in SMC-MR-KO mice compared with Ctl mice (Figure 5c and d).
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L-type Ca2+ channels (Cav1.2) are direct targets of MR in the SMC:15 SMC-MR deletion blunted the activation of L-type Ca2+ channel in mesenteric arteries.15 As CsA was previously shown to increase Ca2+ signaling in the vasculature by increasing the activity of the membrane Cav1.2 channel,23 we hypothesized that the modulation of Cav1.2 activity in the renal microvasculature by MR may contribute to the beneficial effects we observed in SMC-MR-KO mice treated by CsA. We therefore analyzed the effects of KCl (which allows depolarization-induced L-type Ca2+ channel activation) and of BayK8644 (a specific L-type Ca2+ channel agonist) on the renal microvasculature perfusion pressure in isolated kidneys after CsA treatment. Renal arteries from SMC-MR-KO kidneys showed an attenuated contractile response to KCl (Figure 5e) and to BayK8644 (Figure 5f) compared with Ctl kidneys. This demonstrated that in SMC-MR-KO mice, the renal vascular activity of the Ltype Ca2+ channel is blunted after CsA treatment.
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DISCUSSION Our data indicate that the SMC-MR, but not the Endo-MR, is mandatory for acute kidney failure, tubular vacuolization, and NGAL overexpression induced by CsA administration. These effects are associated to a decreased vasoconstrictive response in CsA–treated SMCMR-KO mice that is associated with blunted vascular L-type Ca2+ channel activity.
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The alteration in renal hemodynamics has a pivotal role in the acute nephrotoxicity of transplanted patients treated with CsA as renal vasoconstriction has been established as an initial event linked to acute CIN.4,5,22 In preclinical models, CsA promotes renal afferent arteriolar vasoconstriction3 and increases the vasoactive response to several vasoconstrictors.6–8 Additionally, CsA induces a Ca2+ influx–dependent vasoconstriction in isolated mesenteric arteries,23 and increases the perfusion pressure of the caudal artery.24 This arterial vasoconstriction depends on extracellular Ca2+.23 It has been proposed that CsA enhances signal transmission between G-protein-coupled receptors and L-type Ca2+ channel.24 CsA induces Ca2+ influx in vascular SMC through the IP3 pathway by modulating the phosphorylation of IP3 receptor.6 Interestingly, the use of Ca2+ channel blockers blunts these effects induced by CsA.23–25 In the present study, we showed in CsA– treated mice that SMC-MR deletion prevents: (1) renal microvascular contraction, (2) phosphorylation of contractile proteins in SMC, and (3) vascular L-type Ca2+ channel activity. Both CsA and the MR expressed in vascular SMC affect the Ca2+ signaling pathway, which is essential for SMC contractility. McCurley et al.15 recently demonstrated that vascular SMC-MR modulates the expression of the L-type Ca2+ channel Cav1.2 and that SMC-MR deletion blunts the vasoconstrictive effect of a Cav1.2 agonist. In cardiomyocytes, another contractile cell type where MR has an essential role, MR modulates the activity of both Cav1.2 and the ryanodine receptor, affecting cardiomyocyte contraction.26,27 In the present study, we showed that the effect of SMC-MR deletion on Ltype Ca2+ activity is independent of the abundance of Cav1.2 expression (Supplementary Figure S2a and b online). Furthermore, by modulating cellular Ca2+ homeostasis, SMC-MR will affect downstream signaling to the contractile machinery. Therefore, we propose that vascular SMC-MR, by modulating L-type Ca2+ channel activity and the phosphorylation of proteins critical for SMC contraction, is a key regulator of microvascular contraction and renal hemodynamics after CsA administration. Kidney Int. Author manuscript; available in PMC 2017 February 01.
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CsA administration has been reported to decrease vascular NO production,28 by increasing the synthesis of superoxide anion in renal endothelial cells,29 and decreasing the activity of eNOS,30 catalase, and glutathione peroxidase in kidney.31 These effects lead to endothelial dysfunction and an imbalance in favor of vasoconstriction. Endo-MR-KO has no effect on acute CsA–induced nephrotoxicity, indicating that Endo-MR is not involved in the deleterious endothelial effects of CsA, such as a decrease of vascular eNOS phosphorylation, a surrogate indicator of eNOS activation. MR deletion in SMC did not affect the CsA–induced decrease of vascular eNOS phosphorylation, suggesting that the beneficial effect of MR deletion in vascular SMC is independent of eNOS activity. This is consistent with studies in aortic rings treated with CsA, where endothelium-independent relaxation (induced by sodium nitroprusside) was greater than endothelium-dependent relaxation (induced by acetylcholine),32 suggesting that the overall vascular impact of CsA may be higher in SMC compared with that in the endothelium.
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A key finding of the present study using mice specifically deficient in MR in the vasculature is therefore that SMC-MR deletion is sufficient to protect the kidney from CIN despite intact kidney MR function. This suggests that the vasoconstriction is a critical component of the acute toxicity of CsA on the kidney. We do not exclude that CsA also has a deleterious impact on other cell types such as proximal tubular cells, which are not affected by SMCMR. However, our study supports the idea that renal ischemia caused by CsA–induced vasoconstriction can sensitize the proximal tubule to cell damage induced by CsA. Indeed, we noticed that most of the tubular vacuolization were observed in the pars 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 side effects of CIN and not direct effects on the proximal tubule, where L-type Ca2+ channels have not been reported to be expressed. Previous reports showing the beneficial effects of pharmacological MRAs in experimental models of CIN9–11 could therefore be explained by mitigation of the increased vasoconstrictive response to various vasoactive agents, which are known to be released locally in the renal vasculature after calcineurin inhibition resulting in deleterious renal hemodynamics. Rossi et al.25 showed that part of renal vasoconstriction induced by CsA is related to norepinephrine released from nerve terminals in kidney preparations.25 Indeed, the administration of the a-adrenergic antagonist phenoxybenzamine or renal denervation improved RBF and RVR impairment caused by CsA treatment in conscious rats.34 This vasoconstrictor response elicited by norepinephrine is dependent on L-type Ca2+ channel activity, which could therefore be modulated by SMC-MR.
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Owing to difference in CsA pharmacology in humans and rodents, rodent preclinical models require higher doses of CsA to achieve similar pathology, and these doses are not relevant to human CsA toxicity. Thus, care must be taken when extrapolating from rodent models to human disease. Nevertheless, the impact of CsA on renal hemodynamics in human kidney allograft recipients and the potential benefit of cotreatment with L-type Ca2+ channel antagonists has been highlighted recently by clinical studies. Indeed, daily CsA administration in transplant patients is followed by a transient decrease of the RBF,4 showing a rapid effect of CsA on renal hemodynamics, which in turn can drive the acute
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nephrotoxicity of transplanted patients treated with CsA. It is important to note that the decrease of RBF observed 2 h after CsA treatment each day is attenuated by cotreatment with Ca2+ channel blockers.4 Nankivell et al.35 also demonstrated that CsA decreased RBF (while tacrolimus, another calcineurin inhibitor, did not), an effect mitigated by Ca2+ channel blockade. This finding was also reported in non-hypertensive renal transplant recipients.36 Ca2+ channel blockers were better than both ARB37 and ACE-I38 in improving renal hemodynamics and glomerular filtration rate, independently of beneficial effect on blood pressure control. Ca2+ channel blockers also increased glomerular filtration rate after short-39 and long-40 term use in kidney transplant patients. These clinical data were analyzed in a meta-analysis that showed Ca2+ channel blockers to be the only vasoactive drugs with proven renal benefit during kidney transplantation.41 Interestingly, improved glomerular filtration rate during Ca2+ channel blocker administration was also reported in CsA–treated patients with cardiac transplantation42 and cardiac/lung transplantation.43 Ca2+ channel blockers also demonstrated renal benefits in autoimmune diseases such as psoriasis.44
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Our results show that SMC-MR activation has a crucial role in the pathogenesis of acute CIN by modulating vasoconstriction and increased RVR induced by CsA treatment. These preclinical data strengthen the rationale for the mechanistic roles of both the MR and the Ltype Ca2+ channel and their interaction in acute CsA nephrotoxicity.
MATERIALS AND METHODS Animals
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Genetic ablation of MR in SMC was obtained as described previously,14 whereas deletion in the endothelium was obtained by mating transgenic mice expressing Cre recombinase in endothelial cells (Tie2-Cre mice) with mice harboring MR–floxed alleles,45 resulting in Endo-MR-KO mice. Littermate Cre-negative mice were used as controls. Efficient MR ablation was confirmed in whole aorta from Endo-MR-KO and SMC-MR-KO mice, as compared with Ctl mice, 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: *P < 0.05 Ctl vs. MR-KO). These reductions in the aortic mRNA abundance for MR are similar to those 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 expression was virtually eliminated in SMC from SMCMR-KO aortas when endothelium is removed, (Supplementary Figure S3 online). All animal breeding, housing, and protocols were performed in accordance with the ethical guidelines of INSERM (Institut National de la Santé et de la Recherche Médicale) for the care and use of laboratory animals. Our local ethical committee for animal experimentations at Charles Darwin University approved all experiments under the Ce5/2012/080 record. All animal experimentations adhered to the NIH Guide for Care and Use for the Laboratory Animals. Experimental protocol Eight-week-old C57BL/6 transgenic female mice, with conditional inactivation 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 described
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previously.9 After 1 week, mice were treated with vehicle (EtOH 95% and Cremophor Sigma, in a relation 1:4 (v/v)) or CsA (100mg/kg per day diluted in vehicle solution) subcutaneously during 2 days. This dose of CsA was lower in comparison with the study of Siedlecki et al.,47 but sufficient to induce alterations in renal function and renal structure that mimics the pathological lesions in human acute CIN.17 Of note, the dose of CsA required to induce nephrotoxicity in preclinical rodent models is higher compared with those classically used in human for therapeutic objectives. Biochemical assays At killing, blood was collected in specific tubes and centrifuged to obtain plasma, and then stored at −20 °C. Plasma urea and creatinine were analyzed by an enzymatic method using a Konelab v.7.0.1 automate (Pierce/Thermo Fischer Scientific, Rockford, IL).
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Western blotting
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Abdominal aortas and kidneys were collected and proteins freshly extracted with a sodium dodecyl sulfate 1% buffer pH = 7.4. Proteins were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA), blotted onto nitrocellulose membranes (Amersham ECL Plus; GE Healthcare Life Sciences, Freiburg, Germany), and probed with primary antibodies: anti-NGAL, anti-peNOS, ß-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), antipMLC2, anti-MLC2 (Cell Signaling, Boston, MA), and anti-MR (gift of Prof. Celso GomezSanchez, University of Mississippi Medical Center, Jackson, MS). Secondary antibody antirabbit HRP or anti-mouse HRP (GE Healthcare Life Sciences) was used. Specific binding was detected using enhanced chemiluminescence (Amersham ECL Plus; GE Healthcare Life Sciences) and exposed in a Fujifilm Luminescent Image Analyzer LAS4000 System (Tokyo, Japan). Images of blots were quantified by densitometry analysis (ImageJ 1.43u, US National Institutes of Health, Bethesda, MD). Real-time PCR
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Thoracic aortas were collected and total RNA was extracted with TRIzol Reagent, and cDNA was produced from RNA using Superscript II Reverse Transcriptase Kit (all from Life Technologies Corporation, Carlsbad, CA). Real-time PCR reactions were performed using a Bio-Rad Thermal Cycler (Cergy-St-Christophe, France) (iCycler iQ apparatus) and transcript levels were detected by SYBR Green method. The sequences of the mouse primer pairs are the following: 18S, (F) 5′-CGCCGCTAGAGGTGAAATTC-3′, (R) 5′TCTTGGCAAATGCTTTCGC-3′; MR, (F) 5′-CCAGAAGAGGG GACCACATA-3′, (R) 5′-GGAATTGTCGTAGCCTGCAT-3′; Cav1.2, (F) 5′ATGAAAACACGAGGATGTACGTT-3′, (R) 5′-ACTGACGGTAGAGATGGTTGC-3′. All PCR products were subjected to melting-curve program to confirm amplification specificity. Results were analyzed according to the standard curve method, and mRNA abundance was calculated to the amount of 18S for each sample.
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Histological analysis and immunohistochemistry
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Kidneys were fixed in Bouin solution (Reactifs RAL, Martillac, France) or paraformaldehyde 4% overnight before being included into paraffin blocks. Fourmicrometer sections were performed for hematoxylin–eosin staining (Bouin fixation) and for immunohistochemistry of NGAL. Tubular vacuolization was scored with a semiquantitative score by two pathologists blinded to the experimental groups. Kidney sections were incubated with anti-NGAL antibody (R&D Systems, Minneapolis, MN) before antiimmunoglobulin G secondary antibody for DAB (3,3′-diaminobenzidine) peroxidase revelation. Renal hemodynamics
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After CsA treatment, mice were anesthetized by pentobarbital sodium (50–60 mg/kg body weight intraperitoneally; Nembutal; Abbott, Chicago, IL) and moved to a servo-controlled table kept at 37 °C. The left femoral artery was catheterized for measurement of arterial pressure, and a femoral venous catheter was used for infusion of volume replacement. Bovine serum albumin (4.75 g/dl of saline solution) was infused initially at 50 μl/min to replace surgical losses, and then at 10 μl/min for maintenance. Arterial pressure was measured via a pressure transducer in left femoral artery (Statham P23 DB, Gould, Valley View, OH), and renal blood flow was measured by a flowmeter (0.5v probe; Transonic systems TS420, Ithaca, NY). In vivo RVR was obtained from the relationship between blood pressure and renal blood flow, after intravenous injections of AngII (0.5, 1, and 2 ng; SigmaAldrich). Isolated perfused kidney
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After CsA treatment, the left kidney was isolated and renal artery was cannulated and perfused at 1 ml/min with a Krebs–Henseleit solution (mmol/l: 118.4 NaCl, 4.7 KCl, 2 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 20 NaHCO3, and 11 glucose). The perfusion buffer was maintained at 37 °C and oxygenated (95% O2/5% CO2). After a 30-min equilibration period, the vasoactive response of the renal microvasculature to BayK8644 (0.1 μmol/l; SigmaAldrich) and to increasing concentrations of KCl (0–90 mmol/l) was tested. Changes in perfusion pressures of the renal artery ex vivo were measured with a pressure transducer linked to a PowerLab digital data recorder (PowerLab Chart, ADInstrument-Europe, UK). Statistics
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All data are expressed as the mean ± s.e.m. Data were analyzed by two-way analysis of variance followed by Dunn post hoc test (>2 groups). Mann–Whitney nonparametric test (2 groups), or lineal regression and comparison between slopes for renal hemodynamics. All analyses were performed using GraphPad Prism V6.01 (GraphPad Software, San Diego, CA). P-values
