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Activation of Autophagy and Nucleotide-Binding Domain Leucine-Rich Repeat–Containing-Like Receptor Family, Pyrin Domain–Containing 3 Inflammasome during Leishmania infantum–Associated Glomerulonephritis
2015; Elsevier BV; Volume: 185; Issue: 8 Linguagem: Inglês
10.1016/j.ajpath.2015.04.017
ISSN1525-2191
AutoresKevin J. Esch, Robert G. Schaut, Ian M. Lamb, Gwendolyn Clay, Ádila Lorena Morais Lima, Paulo Ricardo Porfírio do Nascimento, Elizabeth M. Whitley, Selma M. B. Jerônimo, Fayyaz S. Sutterwala, Joseph S. Haynes, Christine A. Petersen,
Tópico(s)Research on Leishmaniasis Studies
ResumoChronic kidney disease is a major contributor to human and companion animal morbidity and mortality. Renal complications are sequelae of canine and human visceral leishmaniasis (VL). Despite the high incidence of infection-mediated glomerulonephritis, little is known about pathogenesis of VL-associated renal disease. Leishmania infantum–infected dogs are a naturally occurring model of VL-associated glomerulonephritis. Membranoproliferative glomerulonephritis type I [24 of 25 (96%)], with interstitial lymphoplasmacytic nephritis [23 of 25 (92%)], and glomerular and interstitial fibrosis [12 of 25 (48%)] were predominant lesions. An ultrastructural evaluation of glomeruli from animals with VL identified mesangial cell proliferation and interposition. Immunohistochemistry demonstrated significant Leishmania antigen, IgG, and C3b deposition in VL dog glomeruli. Asymptomatic and symptomatic dogs had increased glomerular nucleotide-binding domain leucine-rich repeat–containing-like receptor family, pyrin domain containing 3 and autophagosome-associated microtubule-associated protein 1 light chain 3 associated with glomerular lesion severity. Transcriptional analyses from symptomatic dogs confirmed induction of autophagy and inflammasome genes within glomeruli and tubules. On the basis of temporal VL staging, glomerulonephritis was initiated by IgG and complement deposition. This deposition preceded presence of nucleotide-binding domain leucine-rich repeat–containing-like receptor family, pyrin domain containing 3–associated inflammasomes and increased light chain 3 puncta indicative of autophagosomes in glomeruli from dogs with clinical VL and renal failure. These findings indicate potential roles for inflammasome complexes in glomerular damage during VL and autophagy in ensuing cellular responses. Chronic kidney disease is a major contributor to human and companion animal morbidity and mortality. Renal complications are sequelae of canine and human visceral leishmaniasis (VL). Despite the high incidence of infection-mediated glomerulonephritis, little is known about pathogenesis of VL-associated renal disease. Leishmania infantum–infected dogs are a naturally occurring model of VL-associated glomerulonephritis. Membranoproliferative glomerulonephritis type I [24 of 25 (96%)], with interstitial lymphoplasmacytic nephritis [23 of 25 (92%)], and glomerular and interstitial fibrosis [12 of 25 (48%)] were predominant lesions. An ultrastructural evaluation of glomeruli from animals with VL identified mesangial cell proliferation and interposition. Immunohistochemistry demonstrated significant Leishmania antigen, IgG, and C3b deposition in VL dog glomeruli. Asymptomatic and symptomatic dogs had increased glomerular nucleotide-binding domain leucine-rich repeat–containing-like receptor family, pyrin domain containing 3 and autophagosome-associated microtubule-associated protein 1 light chain 3 associated with glomerular lesion severity. Transcriptional analyses from symptomatic dogs confirmed induction of autophagy and inflammasome genes within glomeruli and tubules. On the basis of temporal VL staging, glomerulonephritis was initiated by IgG and complement deposition. This deposition preceded presence of nucleotide-binding domain leucine-rich repeat–containing-like receptor family, pyrin domain containing 3–associated inflammasomes and increased light chain 3 puncta indicative of autophagosomes in glomeruli from dogs with clinical VL and renal failure. These findings indicate potential roles for inflammasome complexes in glomerular damage during VL and autophagy in ensuing cellular responses. Renal disease due to glomerulonephritis and interstitial nephritis is a complication of visceral leishmaniasis (VL), occurring in >96% of symptomatic dogs and 25% to 51% of human cases.1Benderitter T. Casanova P. Nashkidachvili L. Quilici M. Glomerulonephritis in dogs with canine leishmaniasis.Ann Trop Med Parasitol. 1988; 82: 335-341Crossref PubMed Scopus (21) Google Scholar, 2Costa F.A. Goto H. Saldanha L.C. Silva S.M. Sinhorini I.L. Silva T.C. Guerra J.L. Histopathologic patterns of nephropathy in naturally acquired canine visceral leishmaniasis.Vet Pathol. 2003; 40: 677-684Crossref PubMed Scopus (97) Google Scholar, 3Dutra M. Martinelli R. de Carvalho E.M. Rodrigues L.E. Brito E. Rocha H. Renal involvement in visceral leishmaniasis.Am J Kidney Dis. 1985; 6: 22-27Abstract Full Text PDF PubMed Scopus (83) Google Scholar, 4Liborio A.B. Rocha N.A. Oliveira M.J. Franco L.F. Aguiar G.B. Pimentel R.S. Abreu K.L. Silva Jr., G.B. Daher E.F. Acute kidney injury in children with visceral leishmaniasis.Pediatr Infect Dis J. 2012; 31: 451-454Crossref PubMed Scopus (19) Google Scholar Alterations in renal function during active VL in both dogs and humans are usually reversible with prompt anti-Leishmania therapy.5Sayari M. Avizeh R. Barati F. Microscopic evaluation of renal changes in experimental canine visceral leishmaniosis after chemo- and immunotherapy.Pak J Biol Sci. 2008; 11: 1630-1633Crossref PubMed Scopus (2) Google Scholar, 6Beltrame A. Arzese A. Camporese A. Rorato G. Crapis M. Tarabini-Castellani G. Boscutti G. Pizzolitto S. Calianno G. Matteelli A. Di Muccio T. Gramiccia M. Viale P. Acute renal failure due to visceral leishmaniasis by Leishmania infantum successfully treated with a single high dose of liposomal amphotericin B.J Travel Med. 2008; 15: 358-360Crossref PubMed Scopus (13) Google Scholar However, VL-associated kidney disease is progressive and, without therapy, can result in end-stage renal disease (ESRD; approximately 1.5% of human cases).4Liborio A.B. Rocha N.A. Oliveira M.J. Franco L.F. Aguiar G.B. Pimentel R.S. Abreu K.L. Silva Jr., G.B. Daher E.F. Acute kidney injury in children with visceral leishmaniasis.Pediatr Infect Dis J. 2012; 31: 451-454Crossref PubMed Scopus (19) Google Scholar Previous reports regarding the pathophysiology of VL-associated renal disease are conflicting, showing either presence or absence of IgG and complement protein C3.1Benderitter T. Casanova P. Nashkidachvili L. Quilici M. Glomerulonephritis in dogs with canine leishmaniasis.Ann Trop Med Parasitol. 1988; 82: 335-341Crossref PubMed Scopus (21) Google Scholar, 7Costa F.A. Prianti M.G. Silva T.C. Silva S.M. Guerra J.L. Goto H. T cells, adhesion molecules and modulation of apoptosis in visceral leishmaniasis glomerulonephritis.BMC Infect Dis. 2010; 10: 112Crossref PubMed Scopus (27) Google Scholar Accurate assessment of VL-associated glomerular lesions early in their progression is essential to determining an efficacious treatment regimen and aid in determining prognosis. Membranoproliferative glomerulonephritis (MPGN) is traditionally classified as type I, II, or III on the basis of location and character of protein deposits.8Bomback A.S. Appel G.B. Pathogenesis of the C3 glomerulopathies and reclassification of MPGN.Nat Rev Nephrol. 2012; 8: 634-642Crossref PubMed Scopus (116) Google Scholar These characterizations are based on morphology, rather than specific cause, and a spectrum of changes can often be found within a single biopsy specimen. Recent efforts to reclassify MPGN by pathogenesis are better predictors of clinical outcome and may help target therapy. The new classification scheme uses immunofluorescence or immunohistochemistry to define lesions as immune-complex MPGN (with IgG, IgM, and/or complement), as Ig-negative C3 glomerulopathy, or dense deposit disease.8Bomback A.S. Appel G.B. Pathogenesis of the C3 glomerulopathies and reclassification of MPGN.Nat Rev Nephrol. 2012; 8: 634-642Crossref PubMed Scopus (116) Google Scholar, 9Sethi S. Fervenza F.C. Membranoproliferative glomerulonephritis: a new look at an old entity.N Engl J Med. 2012; 366: 1119-1131Crossref PubMed Scopus (347) Google Scholar Podocytes, versatile, long-lived cells constructing the glomerular filtration slit, and mesangial cells, a major resident phagocyte within the glomerular mesangium, play definitive roles in MPGN. The reaction of mesangial cells, ultrastructural changes, and apoptosis of glomerular podocytes are the basis for traditional MPGN classification. The mechanisms through which the glomerular apparatus (podocytes, mesangial cells, and endothelial cells) responds to inflammatory stimuli are not well understood. Multiple reports have demonstrated the importance of autophagy in glomerular basement membrane (GBM) maintenance as a normal function of podocytes.10Hartleben B. Godel M. Meyer-Schwesinger C. Liu S. Ulrich T. Kobler S. Wiech T. Grahammer F. Arnold S.J. Lindenmeyer M.T. Cohen C.D. Pavenstadt H. Kerjaschki D. Mizushima N. Shaw A.S. Walz G. Huber T.B. Autophagy influences glomerular disease susceptibility and maintains podocyte homeostasis in aging mice.J Clin Invest. 2010; 120: 1084-1096Crossref PubMed Scopus (538) Google Scholar, 11Sato S. Yanagihara T. Ghazizadeh M. Ishizaki M. Adachi A. Sasaki Y. Igarashi T. Fukunaga Y. Correlation of autophagy type in podocytes with histopathological diagnosis of IgA nephropathy.Pathobiology. 2009; 76: 221-226Crossref PubMed Scopus (36) Google Scholar, 12Sato S. Adachi A. Sasaki Y. Dai W. Autophagy by podocytes in renal biopsy specimens.J Nippon Med Sch. 2006; 73: 52-53Crossref PubMed Scopus (9) Google Scholar, 13Sato S. Kitamura H. Adachi A. Sasaki Y. Ghazizadeh M. Two types of autophagy in the podocytes in renal biopsy specimens: ultrastructural study.J Submicrosc Cytol Pathol. 2006; 38: 167-174PubMed Google Scholar Podocyte-specific knockout of light chain 3 (LC3), an autophagy protein, resulted in glomerulosclerosis with accumulation of polyubiquitinated proteins by 24 months.10Hartleben B. Godel M. Meyer-Schwesinger C. Liu S. Ulrich T. Kobler S. Wiech T. Grahammer F. Arnold S.J. Lindenmeyer M.T. Cohen C.D. Pavenstadt H. Kerjaschki D. Mizushima N. Shaw A.S. Walz G. Huber T.B. Autophagy influences glomerular disease susceptibility and maintains podocyte homeostasis in aging mice.J Clin Invest. 2010; 120: 1084-1096Crossref PubMed Scopus (538) Google Scholar Renal elevations in LC3 were identified in response to increased glucose in a model of diabetic nephropathy, and in patients with Fabry disease.14Ma T. Zhu J. Chen X. Zha D. Singhal P.C. Ding G. High glucose induces autophagy in podocytes.Exp Cell Res. 2013; 319: 779-789Crossref PubMed Scopus (95) Google Scholar, 15Chevrier M. Brakch N. Celine L. Genty D. Ramdani Y. Moll S. Djavaheri-Mergny M. Brasse-Lagnel C. Annie Laquerriere A.L. Barbey F. Bekri S. Autophagosome maturation is impaired in Fabry disease.Autophagy. 2010; 6: 589-599Crossref PubMed Scopus (79) Google Scholar Regulation of renal fibrosis and the clearance of immune complexes during inflammatory MPGN are potentially regulated through autophagy-associated pathways. Inflammasomes are protein complexes that respond to a variety of cellular stressors and direct receptor-ligand interactions.16Strowig T. Henao-Mejia J. Elinav E. Flavell R. Inflammasomes in health and disease.Nature. 2012; 481: 278-286Crossref PubMed Scopus (1642) Google Scholar The nucleotide-binding domain leucine-rich repeat–containing-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome is well characterized and responds to both pathogen-associated molecular patterns and stress-related molecules.16Strowig T. Henao-Mejia J. Elinav E. Flavell R. Inflammasomes in health and disease.Nature. 2012; 481: 278-286Crossref PubMed Scopus (1642) Google Scholar The NLRP3 inflammasome complex is composed of NLRP3, the adaptor molecule apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), and the cysteine protease caspase-1. NLRP3 responds to a wide array of structurally and chemically diverse agonists, including single- and double-stranded bacterial RNA, ATP, urate crystals, silica, and bacterial pore-forming toxins.17Sutterwala F.S. Haasken S. Cassel S.L. Mechanism of NLRP3 inflammasome activation.Ann N Y Acad Sci. 2014; 1319: 82-95Crossref PubMed Scopus (470) Google Scholar, 18Clay G.M. Sutterwala F.S. Wilson M.E. NLR proteins and parasitic disease.Immunol Res. 2014; 59: 142-152Crossref PubMed Scopus (49) Google Scholar In the kidney, inflammasome products IL-1β and IL-18 are key mediators of renal disease.19Anders H.J. Muruve D.A. The inflammasomes in kidney disease.J Am Soc Nephrol. 2011; 22: 1007-1018Crossref PubMed Scopus (283) Google Scholar Activation of NLRP3-mediated glomerular injury has been shown to be stimulated by NADPH oxidase activation.20Abais J.M. Zhang C. Xia M. Liu Q. Gehr T.W. Boini K.M. Li P.L. NADPH oxidase-mediated triggering of inflammasome activation in mouse podocytes and glomeruli during hyperhomocysteinemia.Antioxid Redox Signal. 2013; 18: 1537-1548Crossref PubMed Scopus (111) Google Scholar This process was recently associated with ASC, a major inflammasome adapter protein, in obesity-induced glomerulonephritis.21Boini K.M. Xia M. Abais J.M. Li G. Pitzer A.L. Gehr T.W. Zhang Y. Li P.L. Activation of inflammasomes in podocyte injury of mice on the high fat diet: effects of ASC gene deletion and silencing.Biochim Biophys Acta. 2014; 1843: 836-845Crossref PubMed Scopus (65) Google Scholar Immune-complex deposition within the subendothelial space and within the glomerular mesangium may drive proinflammatory podocyte and filtration apparatus injury through activation of ASC and NLRP3. Herein, we examined mechanisms that may mediate or be sequelae of MPGN during VL. Glomerulonephritis associated with Leishmania infantum infection was present in both symptomatic and asymptomatic disease and was primarily membranoproliferative, with subendothelial and mesangial electron-dense deposits and endocapillary and mesangial cell hypertrophy. We clearly demonstrated that protein deposits during VL-associated MPGN were composed of L. infantum antigen, IgG, and complement protein C3. This study is the first to evaluate glomerular autophagy and NLRP3 inflammasome presence associated with VL clinical stage in the renal glomerulus. These data suggest that inflammasome engagement and LC3-dependent macroautophagy may contribute to the pathogenesis of immune complex–mediated renal disease and VL-associated MPGN. Animals were staged before necropsy on the basis of clinical presentation, including lymphadenopathy, palpable liver or spleen, and skin lesions typical of VL. Fifteen dogs from the United States were evaluated via full physical examination and fecal examination for comorbidities. The 10 dogs from Brazil, evaluated via full physical examination, were more likely to be symptomatic at the time of presentation (7 of 10 dogs), and also had a higher probability of comorbidities, including malnutrition, helminth infection, or rickettsial infections, and had positive anti-Leishmania antibodies. Study animals were enrolled for this study on the basis of immunofluorescence antibody test serology, as previously described,22Boggiatto P.M. Ramer-Tait A.E. Metz K. Kramer E.E. Gibson-Corley K. Mullin K. Hostetter J.M. Gallup J.M. Jones D.E. Petersen C.A. Immunologic indicators of clinical progression during canine Leishmania infantum infection.Clin Vaccine Immunol. 2010; 17: 267-273Crossref PubMed Scopus (68) Google Scholar and were euthanized with owner's consent. Necropsies were performed by pathologists (K.J.E., J.S.H.) at Iowa State University (Ames, IA) College of Veterinary Medicine and at UFRN (Natal, Brazil). Full or partial necropsies were performed on each animal, with gross evaluation and tissues harvested for histopathological examination. These studies were approved by the Institutional Animal Care and Use Committee at Iowa State University and the Animal Research Ethics Committee of Federal University of Rio Grande do Norte (CEUA-UFRN; Natal, Brazil). Tissues were fixed in 10% neutral-buffered formalin, paraffin embedded, and processed for routine histopathological evaluation. Sections (3 μm thick) were stained with hematoxylin and eosin, Masson's trichrome, or periodic acid– Schiff–methenamine silver. The sections were analyzed by light microscopy (model BX41; Olympus, Center Valley, PA), and renal changes were classified according to World Health Organization criteria for morphological classification glomerulonephritis. Lesions were scored from grade 1 to 4 on the basis of expansion of the mesangium, mesangial fibrosis, thickening of capillary loops, periglomerular inflammation, and percentage of sclerotic glomeruli. All sections were evaluated by a trained veterinary pathologist (K.J.E., J.S.H.). Renal tissue was fixed in 2.0% glutaraldehyde in 0.1 mol/L phosphate buffer, pH 7.4. Ultrathin sections were stained for analysis by transmission electron microscopy. Tissue fragments (1 mm) were fixed in 2.5% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer. After fixation, samples were rinsed in cacodylate buffer, post-fixed in 2% osmium tetroxide, dehydrated in alcohols, cleared in propylene oxide, and embedded in Eponate 12 epoxy resin (PELCO, Redding, CA). Ultrathin sections were cut, stained with uranyl acetate and lead citrate, and examined with a Tecnai 12 G2 electron microscope (FEI, Hillsboro, OR). Formalin-fixed, paraffin-embedded sections (3 μm thick) were labeled with the following: canine anti-L. infantum (hyperimmune serum, 1:500 dilution), anti-canine IgG (2 mg/mL, 1:6000 dilution; Immunovision, Springdale, AZ), anti-canine IgM (2 mg/mL, 1:2000; Immunovision), anti-canine C3b (1 mg/mL, 1:200 dilution; Bethyl Labs, Montgomery, TX). For IgG and IgM labeling, slides were preheated to 57°C for 30 minutes before deparaffinization. Endogenous peroxidases were inhibited by addition of 3% H2O2, followed by a rinse in ultrapure water. Samples were blocked with 10% normal goat serum in phosphate-buffered saline. Samples were stained with multilink (Biogenex, Fremont, CA) and developed with Streptavidin horseradish peroxidase and Nova Red (Vectorlabs, Burlingame, CA) before counterstaining with hematoxylin. Labeling for Leishmania antigen was performed as previously published.23Gibson-Corley K.N. Hostetter J.M. Hostetter S.J. Mullin K. Ramer-Tait A.E. Boggiatto P.M. Petersen C.A. Disseminated Leishmania infantum infection in two sibling foxhounds due to possible vertical transmission.Can Vet J. 2008; 49: 1005-1008PubMed Google Scholar Appropriate controls were included in each experiment. Formalin-fixed, paraffin-embedded sections and cryosections were labeled with 0.2 mg/mL polyclonal canine cross-reactive anti-human LC3 (1:50 dilution; AbCam, Cambridge, MA) and 0.5 mg/mL polyclonal canine cross-reactive anti-human NLRP3 (1:100 dilution; AbCam). Sections were washed and cryosections were fixed with acetone for 5 minutes, then air dried for 30 minutes. Sections were then permeabilized, washed, and treated with blocking immunofluorescence buffer before labeling with primary antibodies. Secondary antibodies for LC3 (Texas Red–conjugated anti-rabbit IgG) and NLRP3 (Alexa Fluor 400–conjugated anti-goat IgG) were used at a concentration of 2 βg/mL. Coverslips were added using Prolong Gold Anti-Fading Reagent (Life Technologies, Grand Island, NY) and were analyzed via confocal microscopy. Secondary antibody only and unstained controls were included for each experiment. A threshold of positivity was generated using color threshold analysis (ImageJ version 1.48; NIH, Bethesda, MD; http://imagej.nih.gov/ij), and immunohistochemical positivity was quantified as area of immunohistochemical-positive staining as a percentage of total glomerular area. For immunofluorescence, glomeruli were imaged individually and analyzed with ImageJ using threshold analysis to calculate percentage of glomerular area with positive labeling, the number of positive cells as a percentage of total glomerular cells was counted as DAPI-positive nuclei in fluorescent images, and density of positive labeling was determined. The Institutional Animal Care and Use Committee at the University of Iowa (Iowa City, IA) approved all mouse protocols used in this study. The generation of Asc−/− mice has been previously described.24Henault J. Martinez J. Riggs J.M. Tian J. Mehta P. Clarke L. Sasai M. Latz E. Brinkmann M.M. Iwasaki A. Coyle A.J. Kolbeck R. Green D.R. Sanjuan M.A. Noncanonical autophagy is required for type I interferon secretion in response to DNA-immune complexes.Immunity. 2012; 37: 986-997Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar Wild-type (WT) C57BL/6N and Asc−/− mice were infected via i.v. injection into the tail vein of 106 L. infantum metacyclic promastigotes. Mice were euthanized 4 weeks after infection, and necropsy was performed in a laminar flow hood. Tissues were collected in 10% neutral-buffered formalin, embedded in Tissue-Tek OCT (Sakura-Finetek USA, Torrance, CA), and frozen as cryosections, or collected in 2.0% glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.4) for transmission electron microscopy, as described. Renal sections were evaluated as described for the dog. No mice in any group had identifiable gross lesions. Overall parasite burdens at 4 weeks after infection, calculated via real-time quantitative PCR (qPCR) on spleen and liver tissues, were highly variable, making statistical evaluation of parasite burden between Asc−/− and WT mice uninformative (data not shown). RNA was isolated via chloroform and column extraction.25Coussens G. Aesaert S. Verelst W. Demeulenaere M. De Buck S. Njuguna E. Inzé D. Van Lijsebettens M. Brachypodium distachyon promoters as efficient building blocks for transgenic research in maize.J Exp Bot. 2012; 63: 4263-4273Crossref PubMed Scopus (42) Google Scholar RNase Out (Life Technologies, Carlsbad, CA) was added to avoid RNase-mediated RNA degradation. Samples were quantified via ND-1000 (Thermo Scientific, Wilmington, DE) to standardize all samples to 200 ng of total RNA per reverse transcription reaction. An iScript cDNA synthesis kit (Bio-Rad, Hercules, CA) was used per manufacturer’s protocol. Reverse transcription was performed in a Vapoprotect thermocycler (Eppendorf, Enfield, CT): 5 minutes at 25°C, 6 minutes at 42°C, 5 minutes at 72°C, and held at 4°C. Primers were designed using the Primer-BLAST Tool (National Center for Biotechnology Information) with the Canis lupus familiaris genome for reference. iTaq Universal SYBR Green Supermix (Bio-Rad) was used for the qPCR with mastermix of primer sets generated. Primers were used at a final concentration of 500 nmol/L per well reaction. An ABI 7000 qPCR machine (Applied Biosystems, Carlsbad, CA) was used. Amplification conditions for all genes were the same: 5 minutes at 95°C, 40 cycles of 15 seconds at 95°C, and 1 minute at 60°C (measure fluorescence step) and a dissociation step of 15 seconds at 95°C, 1 minute at 60°C, 15 seconds at 95°C, and 15 seconds at 60°C. CT values were generated using ABI PRISM SDS Software version 1.2.3 (Applied Biosystems). CT values were calculated and normalized to endogenous control (glyceraldehyde-3-phosphate dehydrogenase) and expressed relative to control dogs using the 2−ΔΔCT method.26Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (123392) Google Scholar Primers are listed in Table 1.Table 1Primers Used for qPCRGene namePrimer sequenceRNA accession no./referenceAtg7Forward: 5′-GGCGGAGGCACCAAATGAT-3′Reverse: 5′-CCACATCCAAGGCACTGCTA-3′XM_005632154.1Lc3Forward: 5′-CGGTACAAGGGTGAGAAGCA-3′Reverse: 5′-GAGCTGTAAGCGCCTCCTAAT-3′XM_536756.3PycardForward: 5′-GAGACCTCACACAAAGGCCA-3′Reverse: 5′-TGCTGGTCCACAAAGTGTGA-3′XM_001003125.1Casp1Forward: 5′-GGCTGTTTGTCCGGTCAGTA-3′Reverse: 5′-CCTCGTGGTTCAGCACTCTT-3′NM_001003125.1Nlrp3Forward: 5′-ACAACAGGGCTGCATCCTAC-3′Reverse: 5′-AGACAATGGTCAGCTCAGGC-3′XM_005623150.1GapdhForward: 5′-CATTGCCCTCAATGACCACT-3′Reverse: 5′-TCCTTGGAGGCCATGTAGAC-3′Maeda et al27Maeda S. Ohno K. Uchida K. Nakashima K. Fukushima K. Tsukamoto A. Nakajima M. Fujino Y. Tsujimoto H. Decreased immunoglobulin A concentrations in feces, duodenum, and peripheral blood mononuclear cells of dogs with inflammatory bowel disease.J Vet Intern Med. 2013; 27: 47-55Crossref PubMed Scopus (34) Google ScholarPrimers were obtained using the Primer-BLAST tool on the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/tools/primer-blast).Atg7, autophagy-related 7; Casp1, IL-1 converting enzyme; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; Lc3, microtubule-associated protein 1 light chain 3β; Nlrp3, nucleotide-binding domain leucine-rich repeat–containing-like receptor family, pyrin domain containing 3; Pycard, N-terminal PYRIN-PAAD-DAPIN domain and a C-terminal caspase-recruitment domain; qPCR, real-time quantitative PCR. Open table in a new tab Primers were obtained using the Primer-BLAST tool on the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/tools/primer-blast). Atg7, autophagy-related 7; Casp1, IL-1 converting enzyme; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; Lc3, microtubule-associated protein 1 light chain 3β; Nlrp3, nucleotide-binding domain leucine-rich repeat–containing-like receptor family, pyrin domain containing 3; Pycard, N-terminal PYRIN-PAAD-DAPIN domain and a C-terminal caspase-recruitment domain; qPCR, real-time quantitative PCR. Archived cryosections were stained with nuclease-free hematoxylin [laser-capture microdissection (LCM) frozen section staining kit, Arcturus Histogene; Applied Biosystems] in ultrapure, nuclease-free water. Sections were dehydrated in alcohol and xylene and prepared for LCM, as previously described.28Sow F.B. Gallup J.M. Sacco R.E. Ackermann M.R. Laser capture microdissection revisited as a tool for transcriptomic analysis: application of an Excel-based qPCR preparation software (PREXCEL-Q).Int J Biomed Sci. 2009; 5: 105-124PubMed Google Scholar Slides were evaluated by a trained pathologist; glomerular tufts, renal proximal tubular epithelium, and renal interstitial regions were excised separately for compartmental expression analysis. Cells were collected onto LCM caps (Capsure; Applied Biosystems) and submerged in 50 βL Tri Reagent (Sigma-Aldrich, St. Louis, MO), and caps were removed by centrifugation. Cells were frozen at −80°C until further processing. RNA extraction, reverse transcription, and qPCR were performed as described above. Cryosections (20 μm thick) were isolated from OCT-embedded dog kidney samples on a cryostat. The cryosections were placed into a mixture of NP-40 lysis buffer and 1× Halt Protease & Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA). The protein concentration of each sample was determined by BCA protein assay kit, according to the manufacturer's instructions (Thermo Fisher Scientific). Lysates were normalized to 5 ng protein per sample, and an equal volume of Laemmli running buffer (1:1 ratio) with 5% β-mercaptoethanol was added. The ASC (anti-mouse/human; AdipoGen, San Diego, CA), NLRP3 (mouse IgG2b anti-mouse/human; AdipoGen), and IL-1β (rabbit polyclonal Ig; Abbiotec, San Diego, CA) were diluted 1:1000 in 5% milk in Tris-buffered saline with 0.05% Tween 20. The secondary antibodies for ASC and IL-1β [Goat Anti-Rabbit IgG (H + L); Jackson Immunoresearch, West Grove, PA] and NLRP3 [Rabbit Anti-Mouse IgG (H + L)-horseradish peroxidase; Southern Biotech, Birmingham, AL] were diluted 1:1000 in Tris-buffered saline with 0.05% Tween 20. SuperSignal West Femto Chemiluminescent Substrate (500 μL; Thermo Fisher Scientific) was added to each membrane; membranes were subsequently imaged by a LI-COR Odyssey (LI-COR Biosciences, Lincoln, NE). Next, membranes were probed for β-actin using an anti–β-actin antibody (mouse monoclonal AC-15; Ambion of Life Technologies, Madison, WI) and a rabbit anti-mouse IgG (H + L)-horseradish peroxidase secondary antibody (Southern Biotech). Quantification of the images obtained was performed using ImageJ software version 1.48 (NIH, Bethesda, MD; http://imagej.nih.gov/ij). Serum samples from four control and four naturally infected dogs were collected from whole blood. As positive control, peripheral blood mononuclear cells were stimulated with lipopolysaccharide for 24 hours, and their lysates were collected. The Canine IL-1β VetSet ELISA Development Kit (Kingfisher Biotech Inc., Saint Paul, MN) was used for all samples, according to the manufacturer’s instructions. Absorbance was measured at 450 nm by a VERSAMAX microplate reader (Molecular Devices, Sunnyvale, CA). Quantification was performed using Microsoft Excel (Redmond, WA). Statistical analysis was conducted with pair-wise t-tests or one-way analysis of variance with Tukey post test, as appropriate (Graph Pad Prism version 5; GraphPad Software, La Jolla, CA); statistical significance was set at α = 0.05. One-way analysis of variance was also performed to demonstrate statistically significant fold changes in transcription data between control and experimental groups. To assess the onset of glomerular lesions and their association with symptomatic VL, we evaluated renal samples from 13 asymptomatic and 12 symptomatic dogs infected with L. infantum, and kidneys from five control (noninfected) dogs that presented to Iowa State University Department of Veterinary Pathology. Predominant microscopic lesions in common to symptomatic and asymptomatic dogs were mesangial hypercellularity (96%; 24 of 25 total cases), glomerular hypersegmentation (96%; 24 of 25 total cases), and interstitial nephritis, characterized by infiltration of macrophages, lymphocytes, and plasma cells into periglomerular and perivascular areas (92%; 23 of 25 total cases) (Table 2 and Figure 1, A and B). Factors predictive of symptomatic disease were irreversible lesions typically associated with chronicity. Glomerular and inter
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