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• W2116098051 abstract "The metabotropic glutamate (mGlu) receptor 1 (GRM1) has been shown to play an important role in neuronal cells by triggering, through calcium release from intracellular stores, various signaling pathways that finally modulate neuron excitability, synaptic plasticity, and mechanisms of feedback regulation of neurotransmitter release. Herein, we show that Grm1 is expressed in glomerular podocytes and that a glomerular phenotype is exhibited by Grm1crv4 mice carrying a spontaneous recessive inactivating mutation of the gene. Homozygous Grm1crv4/crv4 and, to a lesser extent, heterozygous mice show albuminuria, podocyte foot process effacement, and reduced levels of nephrin and other proteins known to contribute to the maintenance of podocyte cell structure. Overall, the present data extend the role of mGlu1 receptor to the glomerular filtration barrier. The regulatory action of mGlu1 receptor in dendritic spine morphology and in the control of glutamate release is well acknowledged in neuronal cells. Analogously, we speculate that mGlu1 receptor may regulate foot process morphology and intercellular signaling in the podocyte. The metabotropic glutamate (mGlu) receptor 1 (GRM1) has been shown to play an important role in neuronal cells by triggering, through calcium release from intracellular stores, various signaling pathways that finally modulate neuron excitability, synaptic plasticity, and mechanisms of feedback regulation of neurotransmitter release. Herein, we show that Grm1 is expressed in glomerular podocytes and that a glomerular phenotype is exhibited by Grm1crv4 mice carrying a spontaneous recessive inactivating mutation of the gene. Homozygous Grm1crv4/crv4 and, to a lesser extent, heterozygous mice show albuminuria, podocyte foot process effacement, and reduced levels of nephrin and other proteins known to contribute to the maintenance of podocyte cell structure. Overall, the present data extend the role of mGlu1 receptor to the glomerular filtration barrier. The regulatory action of mGlu1 receptor in dendritic spine morphology and in the control of glutamate release is well acknowledged in neuronal cells. Analogously, we speculate that mGlu1 receptor may regulate foot process morphology and intercellular signaling in the podocyte. Increasing data provide evidence in favor of the hypothesis that glutamate intercellular signaling in the kidney, mostly driven by podocytes, is relevant to the health of the glomerular filter. Podocytes are highly differentiated cells with a complex ramified structure resembling that of neuronal cells. In common with neurons, podocytes use the same machinery for process formation in such highly arborized structures and possess the necessary vesicular and receptor apparatuses to use glutamatergic transmission.1Rastaldi M.P. Armelloni S. Berra S. Calvaresi N. Corbelli A. Giardino L.A. Li M. Wang G.Q. Fornasieri A. Villa A. Heikkila E. Soliymani R. Boucherot A. Cohen C.D. Kretzler M. Nitsche A. Ripamonti M. Malgaroli A. Pesaresi M. Forloni G.L. Schlondorff D. Holthofer H. D'Amico G. Glomerular podocytes contain neuron-like functional synaptic vesicles.FASEB J. 2006; 20: 976-978Crossref PubMed Scopus (86) Google Scholar, 2Giardino L. Armelloni S. Corbelli A. Mattinzoli D. Zennaro C. Guerrot D. Tourrel F. Ikehata M. Li M. Berra S. Carraro M. Messa P. Rastaldi M.P. Podocyte glutamatergic signaling contributes to the function of the glomerular filtration barrier.J Am Soc Nephrol. 2009; 20: 1929-1940Crossref PubMed Scopus (69) Google Scholar As recently proved, glutamatergic signaling is relevant to the maintenance of glomerular filter integrity because its dysregulation is accompanied by podocyte alterations and increased albuminuria.2Giardino L. Armelloni S. Corbelli A. Mattinzoli D. Zennaro C. Guerrot D. Tourrel F. Ikehata M. Li M. Berra S. Carraro M. Messa P. Rastaldi M.P. Podocyte glutamatergic signaling contributes to the function of the glomerular filtration barrier.J Am Soc Nephrol. 2009; 20: 1929-1940Crossref PubMed Scopus (69) Google Scholar Glutamate is known to be the most abundant excitatory neurotransmitter in the central nervous system. Once released into the synaptic cleft from presynaptic terminals, glutamate can bind to glutamate receptors of two categories: the ionotropic glutamate receptors, which are ligand-gated ion channels that mediate fast excitatory neurotransmission, and the G protein–coupled metabotropic glutamate (mGlu) receptors, which mediate slower, modulatory neurotransmission (reviewed by Olive3Olive M.F. Metabotropic glutamate receptor ligands as potential therapeutics for addiction.Curr Drug Abuse Rev. 2009; 2: 83-98Crossref PubMed Scopus (173) Google Scholar). Three different types of ionotropic glutamate receptors are located on the postsynaptic dendritic spine: the N-methyl-d-aspartate (NMDA) receptor, the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor, and the kainate receptor. The NMDA and AMPA receptors are heterotetrameric protein complexes that regulate the influx of cations (primarily Ca2+ ions) into the neuronal cells. Kainate receptors are tetrameric protein complexes composed of various subunits permeable to Na+ and K+ ions, and, together with the NMDA and AMPA receptors, they contribute to excitatory postsynaptic currents. The mGlu receptors comprise a unique family of at least eight G protein–coupled subtypes (mGlu1 to mGlu8) subdivided into three groups (I, II, and III) and differentiated on the basis of sequence similarity, G protein–coupling specificity, and pharmacologic profile.4Nakanishi S. Molecular diversity of glutamate receptors and implications for brain function.Science. 1992; 258: 597-603Crossref PubMed Scopus (2296) Google Scholar In neurons, the mGlu1 receptor (gene symbol GRM1) has been shown to be expressed at postsynaptic and presynaptic membranes. By releasing calcium from intracellular stores, it triggers various signaling pathways that finally modulate neuron excitability, synaptic plasticity, mechanisms of neuroprotection, and feedback regulation of neurotransmitter release.5Fagni L. Chavis P. Ango F. Bockaert J. Complex interactions between mGluRs, intracellular Ca2+ stores and ion channels in neurons.Trends Neurosci. 2000; 23: 80-88Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 6Nicoletti F. Meek J.L. Iadarola M.J. Chuang D.M. Roth B.L. Costa E. Coupling of inositol phospholipid metabolism with excitatory amino acid recognition sites in rat hippocampus.J Neurochem. 1986; 46: 40-46Crossref PubMed Scopus (352) Google Scholar, 7Rong R. Ahn J.Y. Huang H. Nagata E. Kalman D. Kapp J.A. Tu J. Worley P.F. Snyder S.H. Ye K. PI3 kinase enhancer-Homer complex couples mGluRI to PI3 kinase, preventing neuronal apoptosis.Nat Neurosci. 2003; 6: 1153-1161Crossref PubMed Scopus (246) Google Scholar, 8Ferraguti F. Crepaldi L. Nicoletti F. Metabotropic glutamate 1 receptor: current concepts and perspectives.Pharmacol Rev. 2008; 60: 536-581Crossref PubMed Scopus (167) Google Scholar, 9Gao W. Dunbar R.L. Chen G. Reinert K.C. Oberdick J. Ebner T.J. Optical imaging of long-term depression in the mouse cerebellar cortex in vivo.J Neurosci. 2003; 23: 1859-1866PubMed Google Scholar, 10Hansel C. Linden D.J. D'Angelo E. Beyond parallel fiber LTD: the diversity of synaptic and non-synaptic plasticity in the cerebellum.Nat Neurosci. 2001; 4: 467-475Crossref PubMed Scopus (488) Google Scholar, 11Kano M. Hashimoto K. Tabata T. Type-1 metabotropic glutamate receptor in cerebellar Purkinje cells: a key molecule responsible for long-term depression, endocannabinoid signalling and synapse elimination.Philos Trans R Soc Lond B Biol Sci. 2008; 363: 2173-2186Crossref PubMed Scopus (91) Google Scholar, 12Musante V. Neri E. Feligioni M. Puliti A. Pedrazzi M. Conti V. Usai C. Diaspro A. Ravazzolo R. Henley J.M. Battaglia G. Pittaluga A. Presynaptic mGlu1 and mGlu5 autoreceptors facilitate glutamate exocytosis from mouse cortical nerve endings.Neuropharmacology. 2008; 55: 474-482Crossref PubMed Scopus (48) Google Scholar In particular, the mGlu1 receptor has been shown to play an important role in neuronal cells by regulating the shape of dendritic spines.13Catania M.V. Bellomo M. Di Giorgi-Gerevini V. Seminara G. Giuffrida R. Romeo R. De Blasi A. Nicoletti F. Endogenous activation of group-I metabotropic glutamate receptors is required for differentiation and survival of cerebellar Purkinje cells.J Neurosci. 2001; 21: 7664-7673PubMed Google Scholar At the presynaptic level, it regulates glutamate release.12Musante V. Neri E. Feligioni M. Puliti A. Pedrazzi M. Conti V. Usai C. Diaspro A. Ravazzolo R. Henley J.M. Battaglia G. Pittaluga A. Presynaptic mGlu1 and mGlu5 autoreceptors facilitate glutamate exocytosis from mouse cortical nerve endings.Neuropharmacology. 2008; 55: 474-482Crossref PubMed Scopus (48) Google Scholar The NMDA receptor subunits have also been found to exist on presynaptic terminals,14Garcia-Junco-Clemente P. Linares-Clemente P. Fernandez-Chacon R. Active zones for presynaptic plasticity in the brain.Mol Psychiatry. 2005; 10: 185-200Crossref PubMed Scopus (22) Google Scholar and functional interactions between mGlu1 and NMDA receptors have recently been demonstrated at least at noradrenergic presynaptic terminals, where mGlu1 receptors contribute to the evoked noradrenaline release by enhancing NMDA receptor activity.15Luccini E. Musante V. Neri E. Brambilla Bas M. Severi P. Raiteri M. Pittaluga A. Functional interactions between presynaptic NMDA receptors and metabotropic glutamate receptors co-expressed on rat and human noradrenergic terminals.Br J Pharmacol. 2007; 151: 1087-1094Crossref PubMed Scopus (41) Google Scholar Although most research in the mGlu receptors field has been “synaptically oriented,” recent data extend the role of these receptors to other biological processes. Molecular analyses show functional expression of mGlu receptors in several nonneuronal cells, including bone,16Gu Y. Publicover S.J. Expression of functional metabotropic glutamate receptors in primary cultured rat osteoblasts: cross-talk with N-methyl-d-aspartate receptors.J Biol Chem. 2000; 275: 34252-34259Crossref PubMed Scopus (90) Google Scholar testis,17Storto M. Sallese M. Salvatore L. Poulet R. Condorelli D.F. Dell'Albani P. Marcello M.F. Romeo R. Piomboni P. Barone N. Nicoletti F. De Blasi A. Expression of metabotropic glutamate receptors in the rat and human testis.J Endocrinol. 2001; 170: 71-78Crossref PubMed Scopus (65) Google Scholar and T cells,18Miglio G. Varsaldi F. Dianzani C. Fantozzi R. Lombardi G. Stimulation of group I metabotropic glutamate receptors evokes calcium signals and c-jun and c-fos gene expression in human T cells.Biochem Pharmacol. 2005; 70: 189-199Crossref PubMed Scopus (42) Google Scholar, 19Pacheco R. Ciruela F. Casado V. Mallol J. Gallart T. Lluis C. Franco R. Group I metabotropic glutamate receptors mediate a dual role of glutamate in T cell activation.J Biol Chem. 2004; 279: 33352-33358Crossref PubMed Scopus (112) Google Scholar indicating possible glutamate signaling in different systems (reviewed by Nicoletti and colleagues20Nicoletti F. Battaglia G. Storto M. Ngomba R.T. Iacovelli L. Arcella A. Gradini R. Sale P. Rampello L. De Vita T. Di Marco R. Melchiorri D. Bruno V. Metabotropic glutamate receptors: beyond the regulation of synaptic transmission.Psychoneuroendocrinology. 2007; 32: S40-S45Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). At present, however, no data have been reported, to our knowledge, on a possible role in the kidney. We have been investigating a mouse carrying the spontaneous recessive mutation cervelet-4 (crv4) in the Grm1 gene that causes lack of mGlu1 receptor function and a complex phenotype mainly characterized by ataxia and intention tremor due to impaired cerebellar activities.21Conti V. Aghaie A. Cilli M. Martin N. Caridi G. Musante L. Candiano G. Castagna M. Fairen A. Ravazzolo R. Guenet J.L. Puliti A. crv4, a mouse model for human ataxia associated with kyphoscoliosis caused by an mRNA splicing mutation of the metabotropic glutamate receptor 1 (Grm1).Int J Mol Med. 2006; 18: 593-600PubMed Google Scholar To verify whether mGlu1 receptor could possibly be involved in glomerular function, we first studied its potential expression in the kidney. Then, taking advantage of the availability of Grm1crv4 mice, we analyzed in vivo the renal effects of the lack of functional mGlu1 receptor. The crv4 mutation is a spontaneous recessive mutation that occurs in the BALB/c/Pas inbred strain.21Conti V. Aghaie A. Cilli M. Martin N. Caridi G. Musante L. Candiano G. Castagna M. Fairen A. Ravazzolo R. Guenet J.L. Puliti A. crv4, a mouse model for human ataxia associated with kyphoscoliosis caused by an mRNA splicing mutation of the metabotropic glutamate receptor 1 (Grm1).Int J Mol Med. 2006; 18: 593-600PubMed Google Scholar It consists of a long terminal repeat intronic insertion that disrupts splicing of the mGluR1 gene and causes absence of the protein. Affected Grm1crv4/crv4 and control Grm1+/+ mice are maintained on the same genetic background by intercrossing heterozygous Grm1+/crv4 mice at the Animal Facility of the National Cancer Institute in Genova. All the experimental procedures were performed according to the national current regulations regarding the protection of animals used for scientific purposes (D.L.vo 27/01/1992, n. 116) and were reviewed and approved by the ethical committee for animal experimentation (Comitato per la sperimentazione etica sugli animali). The genotype of the wild-type and Grm1crv4 mice was determined by means of PCR using tail genomic DNA and specific primers as previously reported.12Musante V. Neri E. Feligioni M. Puliti A. Pedrazzi M. Conti V. Usai C. Diaspro A. Ravazzolo R. Henley J.M. Battaglia G. Pittaluga A. Presynaptic mGlu1 and mGlu5 autoreceptors facilitate glutamate exocytosis from mouse cortical nerve endings.Neuropharmacology. 2008; 55: 474-482Crossref PubMed Scopus (48) Google Scholar The human cDNA library (MTC Multiple Tissue cDNA Panels I and II, Clontech Laboratories, Mountain View, CA), 1 μL of cDNA for each tissue sample, was screened by using the GRM1-specific primers Hgrm1F (5′-TGCTGCTGGATTTGCACGGC-3′) and Hgrm1R (5′-TTGCTGCCAGCCAGGATGCG-3′), with 35 PCR amplification cycles. Amplified products obtained from cerebellum and kidney were purified from the agarose gel and were sequenced on both strands using BigDye dideoxy terminator chemistry on an ABI 3100 DNA sequencer (Applied Biosystems, Foster City, CA). Total RNA was extracted from cerebella, renal cortex, sieving-isolated renal glomeruli, and primary cultures of podocytes of wild-type and Grm1crv4/crv4 mice and from an immortalized mouse podocyte cell line and was used to synthesize the first-strand cDNA with gene-specific primers, MGLUR1-R12 (5′-CCTCTCCAGACACTCCAACA-3′) for mouse Grm1 gene, or oligo-dT primers, and the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen, S. Giuliano Milanese, Italy). cDNA amplification was performed using GRM1-specific primers or primers specific for control (nephrin, podocin, and Gapdh) genes: MGLUR1-F3 (5′-TACCCCCAGGCAGGACTAAG-3′) and MGLUR1-R11 (5′-GCATGGTGCATGTTCTGTAGG-3′); MGLUR1-F20 (5′-TCATACGGAAAGGGGAAGTG-3′) and MGLUR1-R24 (5′-CAGCACAAAGATGAGGGTGA-3′); MNephrin-F (5′-AAGCTGGACGTGCATTATGCT-3′) and MNephrin-R (5′-CGGTGCAGACTATATCCACAGAAC-3′); Gapdh-F3 (5′-ATTGTCAGCAATGCATCCTG-3′) and Gapdh-R3 (5′-ATGGACTGTGGTCATGAGCC-3′); and Mpodocin-F1 (5′-TGAAGCGCCTCTTGGCACATCG-3′) and Mpodocin-R2 (5′-TGCAAGTATCGAAGCTGGACAGCG-3′). PCR-specific bands were isolated from the agarose gel and were sequenced. Western blots of cerebella, renal cortex, and sieving-isolated glomeruli were performed according to established protocols21Conti V. Aghaie A. Cilli M. Martin N. Caridi G. Musante L. Candiano G. Castagna M. Fairen A. Ravazzolo R. Guenet J.L. Puliti A. crv4, a mouse model for human ataxia associated with kyphoscoliosis caused by an mRNA splicing mutation of the metabotropic glutamate receptor 1 (Grm1).Int J Mol Med. 2006; 18: 593-600PubMed Google Scholar with some modifications. In brief, cerebella and renal cortex were homogenized in buffer A (5 mmol/L Tris, pH 7.2, 2 mmol/L EDTA, 10 mmol/L iodoacetamide, and protease inhibitors) and then were centrifuged at 30,000 × g for 30 minutes at 4°C to collect a crude membrane pellet. The pellet was lysed in buffer B [20 mmol/L Tris-HCl, pH 6.8, 150 mmol/L NaCl, 10 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100, 10 mmol/L iodoacetamide, and a protease inhibitor mixture (Roche Diagnostics GmbH, Mannheim, Germany)] by quick sonication in short bursts while on ice. Isolated glomeruli were homogenized in lysis buffer (10 mmol/L Tris-HCl, pH 7.5, 10 mmol/L EDTA, 0.5% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, and protease inhibitors) by quick sonication in short bursts while on ice. Protein concentration was determined using the Bradford method (Bio-Rad, Hercules, CA), and samples were separated on 10% gel by means of SDS–polyacrylamide gel electrophoresis. Electroblotted proteins were monitored using Ponceau Red S staining. Membranes were then incubated with the following antibodies: anti-Grm1 monoclonal antibody (1:2500; BD Biosciences, San Jose, CA); guinea pig polyclonal anti-nephrin (GP-N2) (1:1000; PROGEN Biotechnik GmbH, Heidelberg, Germany); rabbit polyclonal anti-podocin (1:2000; Sigma-Aldrich Co., St. Louis, MO); mouse monoclonal anti-synaptopodin (1:500; PROGEN); rabbit polyclonal anti–α-actinin-4 (1:5000; ImmunoGlobe, Himmelstadt, Germany); mouse monoclonal anti–ZO-1 (1:500; Invitrogen); rabbit polyclonal anti-CD2AP (1:1000; Abcam, Cambridge, England); and mouse monoclonal anti-Gapdh (1:10,000; Chemicon, Temecula, CA). After incubation with peroxidase-coupled secondary antibodies, protein bands were detected by using a Western blotting detection system (ECL; Amersham Biosciences, Piscataway, NJ). Bands were detected and analyzed for density using an enhanced chemiluminescence system (Versa-Doc 4000; Bio-Rad), and QuantityOne software (Bio-Rad). For glycosidase treatment, each lysate sample (20 μg) was treated with 1000 U of pNGaseF (New England Biolabs, Ipswich, MA). The reactions were incubated at 37°C for 2 hours, and the samples were analyzed by means of Western blot as reported in the previous paragraph. For primary cultures, kidneys were taken from 7- to 10-day-old Grm1+/+, Grm1+/crv4, and Grm1crv4/crv4 mice as described previously.1Rastaldi M.P. Armelloni S. Berra S. Calvaresi N. Corbelli A. Giardino L.A. Li M. Wang G.Q. Fornasieri A. Villa A. Heikkila E. Soliymani R. Boucherot A. Cohen C.D. Kretzler M. Nitsche A. Ripamonti M. Malgaroli A. Pesaresi M. Forloni G.L. Schlondorff D. Holthofer H. D'Amico G. Glomerular podocytes contain neuron-like functional synaptic vesicles.FASEB J. 2006; 20: 976-978Crossref PubMed Scopus (86) Google Scholar Briefly, glomeruli were isolated by sieving and then were seeded in culture flasks (Corning; Sigma-Aldrich, Milan, Italy) precoated with collagen type IV (Sigma-Aldrich) at 37°C in 5% CO2 atmosphere. After 1 week, first-passage podocytes were separated from glomeruli by an additional sieving through 36-μm mesh. Second-passage podocytes were seeded on flasks to be used for molecular analyses. Some of the cells were instead seeded on Thermanox coverslips (Nunc, VWR International, Milan, Italy) for immunofluorescence studies. Cell characterization was performed using morphologic assessment, immunofluorescence, and Western blot analysis (see Supplemental Figure S1 at http://ajp.amjpathol.org) using podocyte (nephrin and podocin), epithelial (cytokeratins), smooth muscle (α-SMA), and endothelial (CD31) cell markers. The conditionally immortalized mouse podocyte cell line was obtained from transgenic H-2Kb-tsA58 mice, as previously described.2Giardino L. Armelloni S. Corbelli A. Mattinzoli D. Zennaro C. Guerrot D. Tourrel F. Ikehata M. Li M. Berra S. Carraro M. Messa P. Rastaldi M.P. Podocyte glutamatergic signaling contributes to the function of the glomerular filtration barrier.J Am Soc Nephrol. 2009; 20: 1929-1940Crossref PubMed Scopus (69) Google Scholar Cells were first propagated at 33°C in medium containing 20 U/ml of recombinant mouse interferon-γ (Sigma-Aldrich) and then were thermo-shifted to 37°C and maintained in medium without interferon-γ for 15 days. Differentiated cells were used in the experiments. To down-regulate the mGlu1 receptor, differentiated podocytes were incubated in antibiotic-free medium (Opti-MEM; Gibco, Invitrogen, Life Technologies, Carlsbad, CA) for 24 hours and then were transfected with 10- and 20-nM small-interfering RNA (siRNA) duplexes, using Lipofectamine 2000 (Invitrogen, Life Technologies) as the transfection agent. We used a pool of two commercially available siRNAs complementary to Grm1 (Mm01_00091312 and Mm01_00091313; Sigma-Aldrich Co.). As a control, nontargeting siRNAs (Sigma-Aldrich Co.) were applied at the same concentrations. After 24 hours of incubation at 37°C, the medium was replaced with a fresh one containing antibiotics. Transfection efficiency was determined using an siRNA tagged with a fluorescent dye (Alexa Fluor 488; Amersham, PerkinElmer, Waltham, MA), which resulted in higher than 70% efficiency, as estimated from the fluorescence distribution. Silencing of Grm1 and its cellular effects were evaluated 72 hours after siRNA transfection using parallel immunofluorescence and Western blot analyses. Kidneys were taken from 2-, 4-, and 8-month-old animals (3 to 5 Grm1+/+, Grm1+/crv4, and Grm1crv4/crv4 mice, for a total of 30 animals) and were processed for routine light microscopy, transmission electron microscopy, and immunofluorescence studies according to established protocols.22Rastaldi M.P. Armelloni S. Berra S. Li M. Pesaresi M. Poczewski H. Langer B. Kerjaschki D. Henger A. Blattner S.M. Kretzler M. Wanke R. D'Amico G. Glomerular podocytes possess the synaptic vesicle molecule Rab3A and its specific effector rabphilin-3a.Am J Pathol. 2003; 163: 889-899Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar An indirect immunofluorescence method was applied on 5-μm-thick acetone-fixed tissue cryosections and podocyte coverslips. Paraformaldehyde fixation was instead used before revealing F-actin by means of rhodamine-labeled phalloidin (Sigma-Aldrich). The following primary antibodies were used for the study: rabbit anti-Grm1 (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Grm1 antibody (Novus Biologicals, Littleton, CO), rabbit anti-mouse nephrin (intracellular domain) (#035, provided by Harry Holthofer, University of Dublin, Ireland), guinea pig polyclonal anti-nephrin (GP-N2) (PROGEN), mouse anti-synaptopodin (PROGEN), rabbit anti-podocin (Sigma-Aldrich Co.), rabbit anti–ZO-1 (Invitrogen), and rabbit anti-mouse serum albumin (Abcam). As secondary fluorescent-labeled antibodies, we used the following: Alexa Fluor 488 and Alexa Fluor 546 goat anti-rabbit IgG, Alexa Fluor 488 and Alexa Fluor 546 goat anti-mouse IgG highly cross adsorbed, and Alexa Fluor 488 goat anti-guinea pig IgG (Invitrogen). For double stainings, sections/coverslips were first incubated with the first primary antibody followed by the appropriate secondary antibody. After adequate washing, the procedure was repeated for the second primary antibody. Specificity of antibody labeling was demonstrated by the lack of staining after substituting proper control Igs (rabbit primary antibody isotype control and mouse primary antibody isotype control, Invitrogen; and guinea pig primary antibody isotype control, Rockland Immunochemicals, Gilbertsville, PA) for the primary antibodies. Slides were mounted with FluorSave Reagent (Calbiochem, VWR International, Milano, Italy). Images were acquired using a Zeiss Axioscope 40FL microscope equipped with an AxioCam MRc5 digital video camera and immunofluorescence apparatus (Carl Zeiss SpA, Arese, Italy) and were recorded using AxioVision software 4.3. Further images were taken using a confocal laser scanning microscope (Radiance Plus; Bio-Rad, Hemel Hemstead, England) installed on a fluorescence microscope (Eclipse E600; Nikon, Tokyo, Japan). Digital images resulting from the confocal scanning microscopy were optimized for image resolution. Quantitative evaluation was performed on digital images by running appropriate macros, essentially composed using color threshold procedures and filtering, applied on 30 consecutive glomerular areas per specimen selected as the region of interest. The software (AxioVision 4.7 Quantification Modules; Carl Zeiss MicroImaging GmbH, Jena, Germany) was programmed to automatically calculate the percentage of the region of interest occupied by staining, and the results were exported to a spreadsheet file (Microsoft Excel; Microsoft Corp., Redmond, Washington). An indirect immunogold labeling procedure was performed on ultrathin frozen kidney sections, as described previously.22Rastaldi M.P. Armelloni S. Berra S. Li M. Pesaresi M. Poczewski H. Langer B. Kerjaschki D. Henger A. Blattner S.M. Kretzler M. Wanke R. D'Amico G. Glomerular podocytes possess the synaptic vesicle molecule Rab3A and its specific effector rabphilin-3a.Am J Pathol. 2003; 163: 889-899Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar Briefly, after blocking, the material was incubated with the primary rabbit anti-Grm1 antibody followed by the secondary gold-conjugated goat anti-rabbit IgG 12 nm (Jackson ImmunoResearch Europe, Suffolk, England). Specificity of antibody labeling was demonstrated by the lack of staining after substituting a proper control Ig (Invitrogen) for the primary antibody. Three replica spot urine samples per mouse were collected from 3- to 7-month-old Grm1crv4/crv4 (n = 20), Grm1+/crv4 (n = 16), and sex- and age-matched Grm1+/+ (n = 19) mice and centrifuged, and supernatant was conserved at −80°C. Urine albumin levels were measured in duplicate using the indirect competitive enzyme-linked immunosorbent assay Albuwell M kit (Exocell Inc., Philadelphia, PA). Urine creatinine levels were measured in the same samples using Jaffe's reaction, and the urinary albumin excretion (UAE) rate was obtained as the ratio of albumin to creatinine. To measure glomerular permeability to albumin (Palb) in 7-month-old Grm1crv4/crv4, Grm1+/crv4, and wild-type animals, we used the method described by Carraro and colleagues.23Carraro M. Caridi G. Bruschi M. Artero M. Bertelli R. Zennaro C. Musante L. Candiano G. Perfumo F. Ghiggeri G.M. Serum glomerular permeability activity in patients with podocin mutations (NPHS2) and steroid-resistant nephrotic syndrome.J Am Soc Nephrol. 2002; 13: 1946-1952Crossref PubMed Scopus (79) Google Scholar Glomeruli were isolated using standard sieving techniques in medium containing 5 g/dL of bovine serum albumin. Each of 15 to 20 glomeruli per animal were videotaped through an inverted microscope before and after a medium exchange to one containing 10 g/L of bovine serum albumin. The medium exchange created an oncotic gradient across the basement membrane, resulting in a glomerular volume change [ΔV = (Vfinal − Vinitial)/Vinitial], which was measured offline using a video-based image analysis program (SigmaScan Pro; Jandel Scientific Software, Erkrath, Germany). The computer program determines the average radius of the glomerulus in two-dimensional space, and the volume is derived from the formula V = 4/3πr3. The magnitude of ΔV was related to the albumin reflection coefficient (σalb) by the following equation: (σalb)experimental = (ΔV)experimental/(ΔV)control; the σalb of the control glomeruli was assumed to be equal to 1. The Palb was defined as (1 − σalb) and described the movement of albumin subsequent to water flux. When σalb is 0, albumin moves across the membrane with the same velocity as water, and Palb is 1.0; conversely, when σalb is 1.0, albumin cannot cross the membrane with water, and Palb is 0. Podocyte foot process effacement was evaluated according to established protocols.24Deegens J.K. Dijkman H.B. Borm G.F. Steenbergen E.J. van den Berg J.G. Weening J.J. Wetzels J.F. Podocyte foot process effacement as a diagnostic tool in focal segmental glomerulosclerosis.Kidney Int. 2008; 74: 1568-1576Crossref PubMed Scopus (115) Google Scholar To measure mean foot process width (FPW), digital electron micrographs were acquired using a SIS Megaview II camera (Olympus Soft Imaging Solutions GmbH, Munster, Germany) (original magnification, ×7900), and the system was calibrated using the scale bar on the electron micrographs. Six random capillary loops in each of five randomly selected glomeruli per specimen were selected, and the total circumference of the capillary loop was included in the image in 50% of cases. Foot processes were defined as any connected epithelial segment butting on the glomerular basement membrane and separated from the cytoplasmic extensions of the adjacent foot processes by lateral membrane. Only foot processes with lateral membranes clearly identifiable over the entire length were included. The FPW was determined by manually counting the number of foot processes overlying the peripheral capillary basement membrane and measuring basement membrane length using image analysis software. The FPW was calculated as FPW = (π/4)*Σ BML/Σ fp, where Σ fp is the number of foot processes counted on the 30 electron micrographs from each animal, Σ BML is the total peripheral capillary basement membrane length for that animal, and π/4 is a factor used to correct for presumed random variation in the angle of the section relative to the long axis of the foot process. Filtration slit width values were not subtracted; therefore, the reported FPW values represent, on average, the width of one foot process and the adjacent filtration slit. To examine whether the c" @default.
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• W2116098051 title "Albuminuria and Glomerular Damage in Mice Lacking the Metabotropic Glutamate Receptor 1" @default.
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