Innate Immune Cells Are Regulated by Axl in Hypertensive Kidney
2018; Elsevier BV; Volume: 188; Issue: 8 Linguagem: Inglês
10.1016/j.ajpath.2018.04.013
ISSN1525-2191
AutoresSri Nagarjun Batchu, George J. Dugbartey, Kristine M. Wadosky, Deanne Mickelsen, Kyung Ae Ko, Ronald W. Wood, Yuqi Zhao, Xia Yang, Deborah J. Fowell, Vyacheslav A. Korshunov,
Tópico(s)Apelin-related biomedical research
ResumoThe balance between adaptive and innate immunity in kidney damage in salt-dependent hypertension is unclear. We investigated early renal dysfunction and the influence of Axl, a receptor tyrosine kinase, on innate immune response in hypertensive kidney in mice with lymphocyte deficiency (Rag1−/−). The data suggest that increased presence of CD11b+ myeloid cells in the medulla might explain intensified salt and water retention as well as initial hypertensive response in Rag1−/− mice. Global deletion of Axl on Rag1−/− background reversed kidney dysfunction and accumulation of myeloid cells in the kidney medulla. Chimeric mice that lack Axl in innate immune cells (in the absence of lymphocytes) significantly improved kidney function and abolished early hypertensive response. The bioinformatics analyses of Axl-related gene-gene interaction networks established tissue-specific variation in regulatory pathways. It was confirmed that complement C3 is important for Axl-mediated interactions between myeloid and vascular cells in hypertensive kidney. In summary, innate immunity is crucial for renal dysfunction in early hypertension, and is highly influenced by the presence of Axl. The balance between adaptive and innate immunity in kidney damage in salt-dependent hypertension is unclear. We investigated early renal dysfunction and the influence of Axl, a receptor tyrosine kinase, on innate immune response in hypertensive kidney in mice with lymphocyte deficiency (Rag1−/−). The data suggest that increased presence of CD11b+ myeloid cells in the medulla might explain intensified salt and water retention as well as initial hypertensive response in Rag1−/− mice. Global deletion of Axl on Rag1−/− background reversed kidney dysfunction and accumulation of myeloid cells in the kidney medulla. Chimeric mice that lack Axl in innate immune cells (in the absence of lymphocytes) significantly improved kidney function and abolished early hypertensive response. The bioinformatics analyses of Axl-related gene-gene interaction networks established tissue-specific variation in regulatory pathways. It was confirmed that complement C3 is important for Axl-mediated interactions between myeloid and vascular cells in hypertensive kidney. In summary, innate immunity is crucial for renal dysfunction in early hypertension, and is highly influenced by the presence of Axl. The kidney is an important target of hypertension-induced organ injury, because it plays a significant role in the regulation of fluid and electrolyte balance as well as blood pressure (BP). The immune system is proved to regulate angiotensin II (AngII) or deoxycorticosterone-acetate (DOCA) and salt types of hypertension, specifically via T lymphocytes on vascular dysfunction in Rag1−/− mice.1Guzik T.J. Hoch N.E. Brown K.A. 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Spurney R.F. Coffman T.M. Crowley S.D. A novel role for type 1 angiotensin receptors on T lymphocytes to limit target organ damage in hypertension.Circ Res. 2012; 110: 1604-1617Crossref PubMed Scopus (76) Google Scholar Pathophysiological mechanisms involved in the T-lymphocyte–mediated regulation of the kidney and arteries in hypertension are still unclear. Innate immunity [mast cells, eosinophils, basophils, neutrophils, dendritic cells, macrophages, and natural killer (NK) cells] has also been shown to affect kidney function directly or via activation of lymphocytes in hypertension.4Wenzel U. Turner J.E. Krebs C. Kurts C. Harrison D.G. Ehmke H. Immune mechanisms in arterial hypertension.J Am Soc Nephrol. 2016; 27: 677-686Crossref PubMed Scopus (115) Google Scholar The growth arrest–specific gene 6 (Gas6) and its receptor tyrosine kinase (Axl) are known to control innate immune cell functions.5Lemke G. Rothlin C.V. Immunobiology of the TAM receptors.Nat Rev Immunol. 2008; 8: 327-336Crossref PubMed Scopus (531) Google Scholar Gas6 and Axl contribute to DOCA-salt hypertension.6Korshunov V.A. Daul M. Massett M.P. Berk B.C. Axl mediates vascular remodeling induced by deoxycorticosterone acetate salt hypertension.Hypertension. 2007; 50: 1057-1062Crossref PubMed Scopus (30) Google Scholar, 7Park J.K. Theuer S. Kirsch T. Lindschau C. Klinge U. Heuser A. Plehm R. Todiras M. Carmeliet P. Haller H. Luft F.C. Muller D.N. Fiebeler A. Growth arrest specific protein 6 participates in DOCA-induced target-organ damage.Hypertension. 2009; 54: 359-364Crossref PubMed Scopus (14) Google Scholar Axl in bone marrow cells improves early kidney dysfunction, whereas lack of Axl in T cells prevents vascular remodeling in the late phases of DOCA-salt hypertension.8Batchu S.N. Hughson A. Gerloff J. Fowell D.J. Korshunov V.A. Role of Axl in early kidney inflammation and progression of salt-dependent hypertension.Hypertension. 2013; 62: 302-309Crossref PubMed Scopus (19) Google Scholar, 9Batchu S.N. Hughson A. Wadosky K.M. Morrell C.N. Fowell D.J. Korshunov V.A. Role of Axl in T-lymphocyte survival in salt-dependent hypertension.Arterioscler Thromb Vasc Biol. 2016; 36: 1638-1646Crossref PubMed Scopus (9) Google Scholar A complement pathway [complement 3 (C3)] was among one of the major proinflammatory mediators that was down-regulated in chimeras with Axl depletion in bone marrow cells in hypertensive kidney or after vascular injury.8Batchu S.N. Hughson A. Gerloff J. Fowell D.J. Korshunov V.A. Role of Axl in early kidney inflammation and progression of salt-dependent hypertension.Hypertension. 2013; 62: 302-309Crossref PubMed Scopus (19) Google Scholar, 10Gerloff J. Korshunov V.A. Immune modulation of vascular resident cells by Axl orchestrates carotid intima-media thickening.Am J Pathol. 2012; 180: 2134-2143Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar In this study, we focused on exploring the role of innate immunity in early hypertension and the influence of Axl on innate immune cells in hypertensive kidney. Axl wild-type (Axl+/+) and Axl knockout (Axl−/−) male mice were used from our colony. Breeding pairs of B6.129S7-Rag1tm1Mom/J (Rag1−/−) and B6.SJLPtprcaPep3b/BoyJ (B6.SJL) mice and C57BL/6J (B6) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Double wild-type (Axl.Rag1+/+) and double knockout (Axl.Rag1−/−) mice were generated in an intercross between heterozygous Axl.Rag1+/− littermates (Supplemental Figure S1, A and B). Gene presence or deletion was confirmed by two-step genotyping using DNA oligonucleotides (Integrated DNA Technologies, Skokie, IL): Axl (forward: 5′-AGAAGGGGTTAGATGAGG-3′, forward: 5′-ACCGCTTCCTCGTGCTTTA-3′, and reverse: 5′-GCCGAGGTATAGGCTGTC-3′) and Rag1 (forward: 5′-GAGGTTCCGCTACGACTCTG-3′, forward: 5′-TGGATGTGGAATGTGTGCGAG-3′, and reverse: 5′-CCGGACAAGTTTTTCATCGT-3′). Animal facility was controlled by the 12-hour light/dark cycle (lights on at 6 AM, and lights off at 6 PM). Mice had free access to chow and water. The studies were conducted on the basis of guidelines from the NIH and the American Heart Association for the Guide for the Care and Use of Laboratory Animals,11Committee for the Update of the Guide for the Care and Use of Laboratory Animals; National Research Council: Guide for the Care and Use of Laboratory Animals: Eighth Edition. National Academies Press, Washington, DC2011Google Scholar and they were approved by the University of Rochester Animal Care Committee (Rochester, NY). CD4+ T lymphocytes were isolated from spleens and lymph nodes of Axl+/+ mice with CD4+ T-cell enrichment kit (Miltenyi Biotech, Bergisch Gladbach, Germany) with negative magnetic sorting (AutoMACS), as before.9Batchu S.N. Hughson A. Wadosky K.M. Morrell C.N. Fowell D.J. Korshunov V.A. Role of Axl in T-lymphocyte survival in salt-dependent hypertension.Arterioscler Thromb Vasc Biol. 2016; 36: 1638-1646Crossref PubMed Scopus (9) Google Scholar Donor bone marrow cells were collected from tibia and femur bones of the Rag1−/−, Axl.Rag1−/−, and B6.SJL mice, as shown before.8Batchu S.N. Hughson A. Gerloff J. Fowell D.J. Korshunov V.A. Role of Axl in early kidney inflammation and progression of salt-dependent hypertension.Hypertension. 2013; 62: 302-309Crossref PubMed Scopus (19) Google Scholar, 9Batchu S.N. Hughson A. Wadosky K.M. Morrell C.N. Fowell D.J. Korshunov V.A. Role of Axl in T-lymphocyte survival in salt-dependent hypertension.Arterioscler Thromb Vasc Biol. 2016; 36: 1638-1646Crossref PubMed Scopus (9) Google Scholar Peripheral blood or kidneys from mice after adoptive transfer or bone marrow transplant were collected as before.8Batchu S.N. Hughson A. Gerloff J. Fowell D.J. Korshunov V.A. Role of Axl in early kidney inflammation and progression of salt-dependent hypertension.Hypertension. 2013; 62: 302-309Crossref PubMed Scopus (19) Google Scholar, 9Batchu S.N. Hughson A. Wadosky K.M. Morrell C.N. Fowell D.J. Korshunov V.A. Role of Axl in T-lymphocyte survival in salt-dependent hypertension.Arterioscler Thromb Vasc Biol. 2016; 36: 1638-1646Crossref PubMed Scopus (9) Google Scholar Rag1−/− or Axl.Rag1−/− mice were adoptively transferred with donor Axl+/+ CD4+ T cells [6 × 106 in 0.2 mL sterile phosphate-buffered saline (PBS)] via tail vein injections (Supplemental Figure S2). Axl+/+ mice were used as controls. Successful repopulation after 3 weeks was confirmed by flow cytometry with CD4+ antibody in peripheral blood. Bone marrow transplants were done between CD45.2+ donors (Rag1−/− or Axl.Rag1−/− mice) and CD45.1+ recipients (B6.SJL). Recipient mice were irradiated (9.0 Gy) to ablate the host bone marrow in RS2000 irradiator (Rad Source Technologies, Inc., Buford, GA). Within 3 to 4 hours after irradiation, the recipient mice were injected with donor-derived cells (6 × 106 in 0.2 mL sterile PBS) via tail vein. Control chimeras received CD45.1+ BM cells after irradiation (Supplemental Figure S3, A and B). Repopulated cells were confirmed by flow cytometry (CD45.1+, CD45.2+, and CD3+ antibody cocktail) in peripheral blood after 6 weeks. Mouse aortic smooth muscle cells (MASMCs) were isolated from Axl+/+ and Axl−/− mice, as described previously.10Gerloff J. Korshunov V.A. Immune modulation of vascular resident cells by Axl orchestrates carotid intima-media thickening.Am J Pathol. 2012; 180: 2134-2143Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 12Batchu S.N. Xia J. Ko K.A. Doyley M.M. Abe J. Morrell C.N. Korshunov V.A. Axl modulates immune activation of smooth muscle cells in vein graft remodeling.Am J Physiol Heart Circ Physiol. 2015; 309: H1048-H1058Crossref PubMed Scopus (15) Google Scholar MASMCs were used at 70% to 80% confluence of passages from three to five in experiments. MASMC migration was measured using a Boyden chamber assay, as described previously.13Smolock E.M. Korshunov V.A. Pharmacological inhibition of Axl affects smooth muscle cell functions under oxidative stress.Vasc Pharmacol. 2010; 53: 185-192Crossref PubMed Scopus (13) Google Scholar Cells were serum starved with 3% Dulbecco's modified Eagle's medium for 24 hours. Control medium (3% serum Dulbecco's modified Eagle's medium) or medium containing C3 (0.01, 0.1, or 1.0 μg/mL in Dulbecco's modified Eagle's medium) alone or combination with Gas6 (100 nmol/L in Dulbecco's modified Eagle's medium) was placed in the lower chamber. A polyvinylpyrrolidone-free polycarbonate membrane coated with collagen was placed over the bottom wells. Total number of 10,000 cells was placed into the upper chamber and incubated for 6 hours at 37°C in 95% air/5% CO2. Migrated cells to the lower side of the Boyden chamber were fixed and stained using a Diff-Quik staining set (VWR, Radnor, PA). Positive cells were quantified in a blind manner (S.N.B., G.J.D., and V.A.K.) using MCID image software (MCID Elite version 6.0; Imaging Research, Toronto, Canada). Mice were deeply anesthetized, and blood was collected in EDTA-coated collection tubes via heart puncture. Plasma was immediately separated in a centrifuge (Clinical 50; VWR) and stored at −80°C until used as described.12Batchu S.N. Xia J. Ko K.A. Doyley M.M. Abe J. Morrell C.N. Korshunov V.A. Axl modulates immune activation of smooth muscle cells in vein graft remodeling.Am J Physiol Heart Circ Physiol. 2015; 309: H1048-H1058Crossref PubMed Scopus (15) Google Scholar Livers from Axl mice were snap frozen in liquid nitrogen and, later, were sonicated on ice in a cell lysis buffer (1×; Cell Signaling Technology, Danvers, MA) with protease inhibitor (1:1000; Sigma, St. Louis, MO) for 3- to 5-second bursts three times. Homogenates were centrifuged at 8,050 × g for 10 minutes at 4°C (accuSpin Micro17R; Thermo Fisher Scientific, Waltham, MA). Bradford protein assay (Bio-Rad, Hercules, CA) was performed, and equal amounts of protein were separated on 8% SDS gels. Proteins were transferred to nitrocellulose membrane and blocked in 5% milk-PBS for 1 hour at room temperature, followed by overnight incubation in 2% bovine serum albumin–PBS–Tween containing C3 (1:1000; Novus Biologicals, Littleton, CO) and glyceraldehyde-3-phosphate dehydrogenase (1:1000; Cell Signaling Technology) antibodies. Blots were washed in PBS-Tween and incubated with secondary horseradish peroxidase–conjugated rabbit antibodies for 2 hours at room temperature. Protein was visualized using enhanced chemiluminescence. Quantifications of protein levels were assessed through densitometry analyses with ImageJ software version 1.46r (NIH, Bethesda, MD; http://imagej.nih.gov/ij) and expressed as the ratio of the target proteins/loading control. Total C3 level in the plasma from Axl mice was quantified using enzyme-linked immunosorbent assay kit by following manufacturer's instructions (GenWay, San Diego, CA) using a plate reader (Victor2Chan C.T. Sobey C.G. Lieu M. Ferens D. Kett M.M. Diep H. Kim H.A. Krishnan S.M. Lewis C.V. Salimova E. Tipping P. Vinh A. Samuel C.S. Peter K. Guzik T.J. Kyaw T.S. Toh B.H. Bobik A. Drummond G.R. Obligatory role for B cells in the development of angiotensin II-dependent hypertension.Hypertension. 2015; 66: 1023-1033Crossref PubMed Scopus (106) Google Scholar; Wallac, Winooski, VT). Mice were challenged with DOCA-salt mouse model of hypertension that was described before, with minor changes.6Korshunov V.A. Daul M. Massett M.P. Berk B.C. Axl mediates vascular remodeling induced by deoxycorticosterone acetate salt hypertension.Hypertension. 2007; 50: 1057-1062Crossref PubMed Scopus (30) Google Scholar Specifically, mice were anesthetized with an i.p. cocktail of ketamine and xylazine (130 and 9 mg/kg, respectively). An incision was made to expose the left kidney, which was ligated and removed. At the time of surgery, a 75-mg DOCA pellet (21-day release; Innovative Research of America, Sarasota, FL) was placed subcutaneously in a lateral area on the back of mice, and mice were provided with regular chow and 1% NaCl drinking ad libitum. The control animals were uninephrectomized (Nephr) and kept on regular chow and water. To alleviate postoperative pain, mice were treated with i.p. analgesics buprenorphine HCl (0.1 mg/kg) and flunixin meglumine (120 mg/kg). In the pilot studies, it was found that post-surgical pain regimen with opioid analgesic (buprenorphine) with flunixin had no effect on survival and/or development of 6-week DOCA-salt hypertension compared with combination of the flunixin and topical analgesic (bupivacaine, 1 mg/kg) in Axl+/+ mice (Supplemental Figure S4). Systolic BP was measured 1 week after the surgery using noninvasive tail-cuff method plethysmography (Visitech Systems, Apex, NC), as before.6Korshunov V.A. Daul M. Massett M.P. Berk B.C. Axl mediates vascular remodeling induced by deoxycorticosterone acetate salt hypertension.Hypertension. 2007; 50: 1057-1062Crossref PubMed Scopus (30) Google Scholar High-resolution ultrasound system (Vevo2100; FUJIFILM Visual Sonics, Toronto, Canada) was applied to evaluate hypertensive kidneys and renal artery hemodynamics, as previously reported.12Batchu S.N. Xia J. Ko K.A. Doyley M.M. Abe J. Morrell C.N. Korshunov V.A. Axl modulates immune activation of smooth muscle cells in vein graft remodeling.Am J Physiol Heart Circ Physiol. 2015; 309: H1048-H1058Crossref PubMed Scopus (15) Google Scholar Mice were anesthetized with isoflurane and monitored to maintain heart rate >500 beats/min during measurements. Three-dimensional imaging of the right kidneys was captured with the Vevo2100. Kidney volume, vascularity, fluid, and analyzed renal artery hemodynamics were calculated using VevoLab analysis software version 1.6.0 (FUJIFILM VisualSonics, Toronto, Canada). Reconstruction of the kidney vascularity and in-kidney fluid movie (Supplemental Movie S1) were done using Amira 3D software release 5.5 (FEI, Waltham, WA). Mouse urine was collected in Nalgene (North Las Vegas, NV) diuresis cages for 24 hours, as reported.8Batchu S.N. Hughson A. Gerloff J. Fowell D.J. Korshunov V.A. Role of Axl in early kidney inflammation and progression of salt-dependent hypertension.Hypertension. 2013; 62: 302-309Crossref PubMed Scopus (19) Google Scholar Microalbumin Blue 580 assay14Kessler M.A. Meinitzer A. Wolfbeis O.S. Albumin blue 580 fluorescence assay for albumin.Anal Biochem. 1997; 248: 180-182Crossref PubMed Scopus (79) Google Scholar was used, and concentrations of microalbumin were measured in mouse urine. Twenty-four–hour albumin was also calculated in urine volume per body weight (mg/mL per g). The University of Rochester Clinical Laboratories processed samples for Na+ and Cl− (mmol/L) in mouse urine. Na+ and Cl− microequivalents (mEqs) were calculated in urine for 24 hours. Four-color BD Accuri C6 flow cytometer (BD Biosciences, San Jose, CA) was used for detection of the CD4+ T cells (CD4–fluorescein isothiocyanate; eBioscience, Waltham, MA) after adoptive transfer experiments or repopulated cells by cocktail of antibodies (CD45.1–fluorescein isothiocyanate, 1:500; CD45.2-phosphatidylethanolamine (PE), 1:500; and CD3-APC, 1:100; eBioscience) in chimeric mice in peripheral blood. Five major subsets of the immune cells were detected using 12-color LSRII flow cytometer (BD Biosciences), as reported.8Batchu S.N. Hughson A. Gerloff J. Fowell D.J. Korshunov V.A. Role of Axl in early kidney inflammation and progression of salt-dependent hypertension.Hypertension. 2013; 62: 302-309Crossref PubMed Scopus (19) Google Scholar The cells were incubated with a cocktail of CD45.2-PE (1:500; eBioscience), CD3-APC (1:200; eBioscience), CD19-PE-CYC (1:500; eBioscience), CD11b-PE-CY5.5 (1:500; BD Bioscience), CD11c-PE-TXR (1:500; Invitrogen, Carlsbad, CA), and NK1.1-APC-CY7 (1:100; BioLegend, San Diego, CA) antibodies at room temperature for 30 minutes. Cells were washed and resuspended in FACS buffer. Compensation controls were stained with single antibodies. FlowJo software version 7.6.3 (FlowJo LLC, Ashland, OR) was used for flow cytometry analyses. Experimental and control mice were perfusion fixed with 10% paraformaldehyde, and kidneys were processed as before.6Korshunov V.A. Daul M. Massett M.P. Berk B.C. Axl mediates vascular remodeling induced by deoxycorticosterone acetate salt hypertension.Hypertension. 2007; 50: 1057-1062Crossref PubMed Scopus (30) Google Scholar, 8Batchu S.N. Hughson A. Gerloff J. Fowell D.J. Korshunov V.A. Role of Axl in early kidney inflammation and progression of salt-dependent hypertension.Hypertension. 2013; 62: 302-309Crossref PubMed Scopus (19) Google Scholar Kidney cross sections were incubated with 3% H2O2 to block endogenous peroxidase activity. Antigen retrieval was performed with a Decloaker buffer (pH = 6.0; Biocare, Pacheco, CA), and high temperature was applied in the following protocols. A rat anti-mouse Mac-2 (1:15,000; Cedarlane, Burlington, ON, Canada) antibody was applied for 60 minutes at room temperature, which was subsequently stained with rabbit anti-mouse T-cell Ig and mucin domain 1 (TIM-1; 1:5000; Thermo Fisher Scientific) overnight at 4°C. A mouse anti-mouse CD45.2 (1:100; Pharmingen, San Jose, CA) antibody was applied at 37°C for 60 minutes, which was followed by rabbit anti-mouse TIM-1 (1:5000; Thermo Fisher Scientific) overnight at 4°C. A rabbit anti-mouse C3 (1:2000; Novus Biologicals) was incubated overnight at 4°C. A rabbit anti-Axl (1:500; Abcam, Cambridge, UK) antibody was incubated for 60 minutes at room temperature. Peroxidase-binding sites (Mac-2, CD45.2, C3, and Axl) were verified with 3,3′-diaminobenzidine (Dako, Santa Clara, CA) after application of the rat-on-mouse, mouse-on-mouse, or rabbit-on-mouse horseradish peroxidase–polymer (Biocare). The alkaline-phosphatase–binding sites (TIM-1) were recognized by Fast Red (Vulcan Red; Biocare) after rabbit-on-rodent or mouse-on-mouse alkaline-phosphatase-polymer (Biocare). All double-labeled slides were counterstained with methyl green or hematoxylin, as before.9Batchu S.N. Hughson A. Wadosky K.M. Morrell C.N. Fowell D.J. Korshunov V.A. 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