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• W2031389630 abstract "The atypical protein kinase C (aPKC) isotypes PKCλ/ι and PKCζ are both expressed in podocytes; however, little is known about differences in their function. Previous studies in mice have demonstrated that podocyte-specific loss of PKCλ/ι leads to a severe glomerular phenotype, whereas mice deficient in PKCζ develop no renal phenotype. We analyzed various effects caused by PKCλ/ι and PKCζ deficiency in cultured murine podocytes. In contrast to PKCζ-deficient podocytes, PKCλ/ι-deficient podocytes exhibited a severe actin cytoskeletal phenotype, reduced cell size, decreased number of focal adhesions, and increased activation of small GTPases. Comparative microarray analysis revealed that the guanine nucleotide exchange factor Def-6 was specifically up-regulated in PKCλ/ι-deficient podocytes. In vivo Def-6 expression is significantly increased in podocytes of PKCλ/ι-deficient mice. Cultured PKCλ/ι-deficient podocytes exhibited an enhanced membrane association of Def-6, indicating enhanced activation. Overexpression of aPKCλ/ι in PKCλ/ι-deficient podocytes could reduce the membrane-associated expression of Def-6 and rescue the actin phenotype. In the present study, PKCλ/ι was identified as an important factor for actin cytoskeletal regulation in podocytes and Def-6 as a specific downstream target of PKCλ/ι that regulates the activity of small GTPases and subsequently the actin cytoskeleton of podocytes. The atypical protein kinase C (aPKC) isotypes PKCλ/ι and PKCζ are both expressed in podocytes; however, little is known about differences in their function. Previous studies in mice have demonstrated that podocyte-specific loss of PKCλ/ι leads to a severe glomerular phenotype, whereas mice deficient in PKCζ develop no renal phenotype. We analyzed various effects caused by PKCλ/ι and PKCζ deficiency in cultured murine podocytes. In contrast to PKCζ-deficient podocytes, PKCλ/ι-deficient podocytes exhibited a severe actin cytoskeletal phenotype, reduced cell size, decreased number of focal adhesions, and increased activation of small GTPases. Comparative microarray analysis revealed that the guanine nucleotide exchange factor Def-6 was specifically up-regulated in PKCλ/ι-deficient podocytes. In vivo Def-6 expression is significantly increased in podocytes of PKCλ/ι-deficient mice. Cultured PKCλ/ι-deficient podocytes exhibited an enhanced membrane association of Def-6, indicating enhanced activation. Overexpression of aPKCλ/ι in PKCλ/ι-deficient podocytes could reduce the membrane-associated expression of Def-6 and rescue the actin phenotype. In the present study, PKCλ/ι was identified as an important factor for actin cytoskeletal regulation in podocytes and Def-6 as a specific downstream target of PKCλ/ι that regulates the activity of small GTPases and subsequently the actin cytoskeleton of podocytes. The protein kinase C (PKC) family consists of 10 serine/threonine kinases, which can be divided into three subfamilies on the basis of their different regulatory domains. Conventional PKCs (PKCα, PKCβI, PKCβII, and PKCγ) and novel PKCs (PKCδ, PKCθ, PKCη, and PKCε) can be activated by calcium or diacylglycerol (DAG) via C1 and C2 domains. The third subfamily, atypical PKCs (aPKCs) [PKCλ/ι (PKCλ in mouse and PKCι in human) and PKCζ], lack the calcium-sensitive C2 domain and possess an atypical C1 domain that does not bind DAG but binds phosphatidylinositol 3,4,5-triphosphate (PIP3) or ceramide instead.1Steinberg S.F. Structural basis of protein kinase C isoform function.Physiol Rev. 2008; 88: 1341-1378Crossref PubMed Scopus (644) Google Scholar The amino acid sequences of PKCλ/ι and PKCζ are 72% identical2Selbie L.A. Schmitz-Peiffer C. Sheng Y. Biden T.J. Molecular cloning and characterization of PKC iota, an atypical isoform of protein kinase C derived from insulin-secreting cells.J Biol Chem. 1993; 268: 24296-24302Abstract Full Text PDF PubMed Google Scholar; however, the functions of these two proteins in vivo seem to be distinct. Because complete knockout of PKCλ/ι in mice is lethal before embryonic day 9,3Soloff R.S. Katayama C. Lin M.Y. Feramisco J.R. Hedrick S.M. Targeted deletion of protein kinase C lambda reveals a distribution of functions between the two atypical protein kinase C isoforms.J Immunol. 2004; 173: 3250-3260Crossref PubMed Scopus (73) Google Scholar we created a podocyte-specific PKCλ/ι knockout (PKCλ/ι−/−) mouse by mating mice with a floxed PKCλ/ι gene and Podocin-Cre mice4Moeller M.J. Sanden S.K. Soofi A. Wiggins R.C. Holzman L.B. Podocyte-specific expression of cre recombinase in transgenic mice.Genesis. 2003; 35: 39-42Crossref PubMed Scopus (249) Google Scholar with expression of the Cre recombinase dependent on the podocyte-specific podocin promoter. Podocyte-specific deletion of PKCλ/ι led to a severe glomerular phenotype with glomerulosclerosis, proteinuria, and death at age 4 to 5 weeks.5Huber T.B. Hartleben B. Winkelmann K. Schneider L. Becker J.U. Leitges M. Walz G. Haller H. Schiffer M. Loss of podocyte aPKClambda/iota causes polarity defects and nephrotic syndrome.J Am Soc Nephrol. 2009; 20: 798-806Crossref PubMed Scopus (81) Google Scholar In contrast to PKCλ/ι, complete knockout of PKCζ (PKCζ−/−) is rather mild. PKCζ−/− mice develop no obvious renal phenotype but exhibit changes in their secondary lymphatic organs as a result of a disturbed NF-κB pathway.6Leitges M. Sanz L. Martin P. Duran A. Braun U. Garcia J.F. Camacho F. Diaz-Meco M.T. Rennert P.D. Moscat J. Targeted disruption of the zetaPKC gene results in the impairment of the NF-kappaB pathway.Mol Cell. 2001; 8: 771-780Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar Recently, we showed that double-knockout of aPKCs in podocytes leads to a developmental phenotype with no formation of a normal foot processes network, defective glomerular maturation, incomplete capillary formation, and mesangiolysis.7Hartleben B. Widmeier E. Suhm M. Worthmann K. Schell C. Helmstadter M. Wiech T. Walz G. Leitges M. Schiffer M. Huber T.B. aPKCλ/ι and aPKCζ contribute to podocyte differentiation and glomerular maturation.J Am Soc Nephrol. 2013; 24: 253-267Crossref PubMed Scopus (31) Google Scholar The severe podocyte foot process effacement in the podocyte-specific PKCλ/ι−/− mice and the inability to form foot processes in the double-knockout mice suggests an important role for the actin cytoskeleton of podocytes. The actin cytoskeleton of podocytes is a highly dynamic and complex structure8Andrews P.M. Bates S.B. Filamentous actin bundles in the kidney.Anat Rec. 1984; 210: 1-9Crossref PubMed Scopus (34) Google Scholar, 9Vasmant D. Maurice M. Feldmann G. Cytoskeleton ultrastructure of podocytes and glomerular endothelial cells in man and in the rat.Anat Rec. 1984; 210: 17-24Crossref PubMed Scopus (58) Google Scholar, 10Ichimura K. Kurihara H. Sakai T. Actin filament organization of foot processes in rat podocytes.J Histochem Cytochem. 2003; 51: 1589-1600Crossref PubMed Scopus (109) Google Scholar, 11Kriz W. Mundel P. Elger M. The contractile apparatus of podocytes is arranged to counteract GBM expansion.Contrib Nephrol. 1994; 107: 1-9Crossref PubMed Google Scholar that also has an important role in maintenance of the slit diaphragm in various renal diseases.12Pavenstädt H. Kriz W. Kretzler M. Cell biology of the glomerular podocyte.Physiol Rev. 2003; 83: 253-307Crossref PubMed Scopus (1203) Google Scholar When the actin cytoskeleton of podocyte foot processes changes from parallel bundles of actin13Drenckhahn D. Franke R.P. Ultrastructural organization of contractile and cytoskeletal proteins in glomerular podocytes of chicken, rat, and man.Lab Invest. 1988; 59: 673-682PubMed Google Scholar to a dense network of short filaments, foot process effacement and, in turn, proteinuria can develop.14Kerjaschki D. Caught flat-footed: podocyte damage and the molecular bases of focal glomerulosclerosis.J Clin Invest. 2001; 108: 1583-1587Crossref PubMed Scopus (266) Google Scholar A group of key regulators for actin cytoskeletal regulation are the small GTPases, which are molecular switches that change between a GDP-bound inactive form and a GTP-bound active form. Guanine nucleotide exchange factors (GEFs) control the GDP/GTP exchange by increasing the exchange rate, which leads to activation of small GTPases.15Hall A. Rho GTPases and the actin cytoskeleton.Science. 1998; 279: 509-514Crossref PubMed Scopus (5216) Google Scholar Recent studies have demonstrated the critical involvement of small GTPases in regulation of podocyte actin cytoskeleton in normal podocyte homeostasis and in acquired and genetic glomerular diseases.16Akilesh S. Suleiman H. Yu H. Stander M.C. Lavin P. Gbadegesin R. Antignac C. Pollak M. Kopp J.B. Winn M.P. Shaw A.S. Arhgap24 inactivates Rac1 in mouse podocytes, and a mutant form is associated with familial focal segmental glomerulosclerosis.J Clin Invest. 2011; 121: 4127-4137Crossref PubMed Scopus (202) Google Scholar, 17Wang L. Ellis M.J. Gomez J.A. Eisner W. Fennell W. Howell D.N. Ruiz P. Fields T.A. Spurney R.F. Mechanisms of the proteinuria induced by Rho GTPases.Kidney Int. 2012; 81: 1075-1085Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 18van Duijn T.J. Anthony E.C. Hensbergen P.J. Deelder A.M. Hordijk P.L. Rac1 recruits the adapter protein CMS/CD2AP to cell-cell contacts.J Biol Chem. 2010; 285: 20137-20146Crossref PubMed Scopus (43) Google Scholar, 19Zhu L. Jiang R. Aoudjit L. Jones N. Takano T. Activation of RhoA in podocytes induces focal segmental glomerulosclerosis.J Am Soc Nephrol. 2011; 22: 1621-1630Crossref PubMed Scopus (104) Google Scholar Herein we demonstrate that differentially expressed FDCP-6 (Def-6), a GEF for the Rho GTPases Rac1 and Cdc42,20Gupta S. Fanzo J.C. Hu C. Cox D. Jang S.Y. Lee A.E. Greenberg S. Pernis A.B. T cell receptor engagement leads to the recruitment of IBP, a novel guanine nucleotide exchange factor, to the immunological synapse.J Biol Chem. 2003; 278: 43541-43549Crossref PubMed Scopus (66) Google Scholar is differentially affected by the aPKC isoforms λ/ι and ζ in podocytes. Def-6, also known as swap70-like adaptor of T cells or IRF-4–binding protein, consists of an EF-hand motif at the N terminus, a pleckstrin homology domain, and a Dbl homology–like domain (DHL) at its C terminus. Def-6 contains 631 amino acids, has a predicted molecular weight of 74 kD,21Gupta S. Lee A. Hu C. Fanzo J. Goldberg I. Cattoretti G. Pernis A.B. Molecular cloning of IBP, a SWAP-70 homologous GEF, which is highly expressed in the immune system.Hum Immunol. 2003; 64: 389-401Crossref PubMed Scopus (69) Google Scholar and exhibits much homology to the B cell–specific GEF swap70. To date, most investigations of the function or expression of Def-6 have been performed in B or T cells because Def-6 is highly expressed in lymphatic organs.21Gupta S. Lee A. Hu C. Fanzo J. Goldberg I. Cattoretti G. Pernis A.B. Molecular cloning of IBP, a SWAP-70 homologous GEF, which is highly expressed in the immune system.Hum Immunol. 2003; 64: 389-401Crossref PubMed Scopus (69) Google Scholar Herein we report for the first time an important role for Def-6 in a renal epithelial cell type. The plasmids Def-6 FL (bp 1–2015 in pEGFP-C2) and Def-6 DHL (bp 1066–1934 in pEGFP-C3) have been used in previous studies.22Mavrakis K.J. McKinlay K.J. Jones P. Sablitzky F. DEF6, a novel PH-DH-like domain protein, is an upstream activator of the Rho GTPases Rac1, Cdc42, and RhoA.Exp Cell Res. 2004; 294: 335-344Crossref PubMed Scopus (34) Google Scholar Primary antibodies used were mouse monoclonal anti-vinculin (clone hVIN-1) (Sigma-Aldrich Corp., St. Louis, MO), mouse anti-CD29 (integrin β1) (BD Transduction Laboratories, BD Biosciences, San Jose, CA), goat anti-podocalyxin (R&D Systems GmbH, Wiesbaden, Germany), mouse monoclonal anti-RhoA, mouse monoclonal anti-Rac1, mouse monoclonal anti-Cdc42 (Cytoskeleton, Inc., Denver, CO), mouse monoclonal anti–Def-6 (Abnova Corp., Taipei, Taiwan), affinity purified rabbit anti–Def-6 (according to a previous study23Samson T. Will C. Knoblauch A. Sharek L. von der Mark K. Burridge K. Wixler V. Def-6, a guanine nucleotide exchange factor for Rac1, interacts with the skeletal muscle integrin chain alpha7A and influences myoblast differentiation.J Biol Chem. 2007; 282: 15730-15742Crossref PubMed Scopus (27) Google Scholar), rabbit antiserum anti-Giα3 (Upstate Biotechnology, Lake Placid, NY), rabbit polyclonal anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), rabbit polyclonal anti-Sp1 (Santa Cruz Biotechnology), rat monoclonal anti-entactin/nidogen (Millipore Corp., Billerica, MA), and rabbit monoclonal anti-PKCλ/ι (Cell Signaling Technology, Inc., Beverly, MA). Polyclonal antibody specific for PKCζ was raised in rabbits and has been described previously.24Helfrich I. Schmitz A. Zigrino P. Michels C. Haase I. le Bivic A. Leitges M. Niessen C.M. Role of aPKC isoforms and their binding partners Par3 and Par6 in epidermal barrier formation.J Invest Dermatol. 2007; 127: 782-791Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar All secondary antibodies used for immunostaining were obtained from Invitrogen Corp. (Carlsbad, CA) and included rabbit–Alexa Fluor 488, mouse–Alexa Fluor 488, goat–Alexa Fluor 488, rabbit–Alexa Fluor 555, mouse–Alexa Fluor 555, and rat–Alexa Fluor 488. All secondary antibodies used for Western blotting were obtained from Santa Cruz Biotechnology and included goat anti-mouse IgG–horseradish peroxidase and goat anti-rabbit IgG horseradish peroxidase. Further reagents included Alexa Fluor 546 phalloidin (Invitrogen, Darmstadt, Germany), PKCι siRNA, control siRNA (Santa Cruz Biotechnology), aPKC pseudosubstrate and scrambled peptide (BioSource International, Inc., Camarillo, CA), and Def6 (human) recombinant protein (Abnova). Generation of podocyte-specific aPKCλ/ι knockout mice using floxed PKCλ/ι and Podocin-Cre mice has been described previously.5Huber T.B. Hartleben B. Winkelmann K. Schneider L. Becker J.U. Leitges M. Walz G. Haller H. Schiffer M. Loss of podocyte aPKClambda/iota causes polarity defects and nephrotic syndrome.J Am Soc Nephrol. 2009; 20: 798-806Crossref PubMed Scopus (81) Google Scholar Podocyte-specific aPKCλ/ι heterozygous mice were crossed with Immorto transgenic mice.25Jat P.S. Noble M.D. Ataliotis P. Tanaka Y. Yannoutsos N. Larsen L. Kioussis D. Direct derivation of conditionally immortal cell lines from an H-2Kb-tsA58 transgenic mouse.Proc Natl Acad Sci U S A. 1991; 88: 5096-5100Crossref PubMed Scopus (628) Google Scholar Homozygous podocyte-specific aPKCλ/ι mice harboring the Immorto transgene were used for generation of immortalized podocyte cell lines. Glomeruli were isolated from kidneys of 4-week-old mice using a sequential sieving technique as described previously.26Tossidou I. Starker G. Kruger J. Meier M. Leitges M. Haller H. Schiffer M. PKC-alpha modulates TGF-beta signaling and impairs podocyte survival.Cell Physiol Biochem. 2009; 24: 627-634Crossref PubMed Scopus (40) Google Scholar Only stable cell lines derived from these glomeruli that were positive for the podocyte-specific markers WT-1 and synaptopodin were used in the experiments. Immortalized murine podocytes were cultured as described by Mundel et al.27Mundel P. Reiser J. Zúñiga Mejía B.A. Pavenstädt H. Davidson G.R. Kriz W. Zeller R. Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines.Exp Cell Res. 1997; 236: 248-258Crossref PubMed Scopus (765) Google Scholar In brief, cells were cultured on collagen I–coated flasks using RPMI 1640 medium containing 10% fetal calf serum, 1% penicillin/streptomycin, and 10 U/mL γ-interferon at 33°C for proliferation. For differentiation, cells were cultured at 37°C without γ-interferon for 10 to 14 days. For stimulation experiments, cells were serum starved with 1% fetal calf serum overnight and then treated with 10 μmol/L aPKC pseudosubstrate for 2 hours at 37°C or 10 μmol/L scrambled peptide, respectively. Differentiated deficient and control cells were observed under a microscope (Axio Observer.Z1; Zeiss Microscopy GmbH, Göttingen, Germany) at ×20 magnification for 6 hours. Images were obtained automatically every 2 minutes. To quantify the movement of cells, all images were imported into ImageJ software version 1.44p. Using the Manual Tracking plug-in, the nucleus of every selected cell (10 cells of each genotype) was marked on the image sequence. The program measures the distance between two frames, and by summing all distances, the distance covered from the start of recording to the end, at 6 hours. The day before transfection, podocytes were seeded on glass coverslips and cultured at 33°C overnight. Cells were transfected using FuGENE HD transfection reagent (Roche Diagnostics GmbH, Mannheim, Germany) according to the protocol recommended by the manufacturer. Transfection with PKCλ/ι siRNA or control siRNA (Santa Cruz Biotechnology) was performed on 3-day differentiated podocytes using lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s protocol. After 6 hours in the incubator, the medium was changed to normal growth medium. After transfection, cells were cultured for 72 hours at 37°C and fixed with 4% paraformaldehyde. Plasmids for adenovirus production were generated using Gateway technology (Invitrogen). Donor vectors were generated by amplifying Green fluorescent protein (GFP)–tagged full-length human PKCι or GFP alone with specific primers and cloning into the pDONR221 vector by using BP clonase II enzyme for recombination. Adenoviral constructs were generated using LR Clonase II enzyme, which recombines pDONR221 with pAd/CMV/V5-DEST. All reactions were performed as recommended by the manufacturer (Invitrogen). Adenoviral expression and amplification were performed in HEK293 cells. Podocytes were infected with equal amounts of PKCι-GFP or GFP adenoviral supernatants. After 7 days, podocytes were either harvested for RNA isolation or fixed with 4% paraformaldehyde for immunostaining. Genomic DNA from cultured podocytes was prepared using the basicdna-OLS Kit (OMNI Life Science GmbH & Co. KG, Hamburg, Germany) following the protocol recommended by the manufacturer. As controls, genomic DNA samples from tail biopsies were used. PCR was performed under standard conditions in a Primus Thermocycler (MWG-Biotech AG, Ebersberg, Germany) with the following primer pairs: Cre forward, 5′-AGGTTCGTTCACTCATGGA-3′; Cre reverse, 5′-TGCACCAGTTTAGTTACCC-3′; loxP forward, 5′-TTGTGAAAGCGACTGGATTG-3′; loxP reverse, 5′-CTTGGGTGGAGAGGCTATTC-3′; WT reverse, 5′-AATGTTCATGTTCAACACTGCT-3′; Highzeta5 forward, 5′-GCCATCTCCAACAGCCACAG-3′; Highzeta5ex, 5′-CCGTTGGCTCGGTACAGCTT-3′; and MO13, 5′-CTTGGGTGGAGAGGCTATTC-3′. Immortalized cultured mouse podocytes were seeded on glass coverslips and differentiated for 10 to 14 days. After fixation with 4% paraformaldehyde, cells were permeabilized with 0.1% Triton X-100, blocked in 10% normal donkey serum, and incubated overnight at 4°C with the primary antibodies as indicated. After rinsing with PBS, the cells were incubated with a fluorophore-conjugated secondary antibody for 1 hour and mounted in medium containing DAPI. For immunofluorescence staining of frozen kidneys, 6-μm sections (Leica CM3050S cryostat; Leica Microsystems, Wetzlar, Germany) were fixed in acetone, blocked with 10% normal donkey serum, and incubated with the primary antibodies, as indicated, overnight at 4°C. After rinsing with PBS, sections were incubated with conjugated secondary antibodies for 1 hour at room temperature and mounted in medium containing DAPI. Confocal images were obtained using a Leica Inverted-2 microscope with a 63× oil immersion objective. After 10 to 14 days of differentiation, cells were lyzed on ice in radioimmunoprecipitation assay buffer [50 mmol/L Tris (pH 7.5), 150 mmol/L NaCl 0.5% sodium deoxycholate, 1% nonidet P-40, and 0.1% SDS] supplemented with protease inhibitors (cOmplete, Mini; Roche Diagnostics), 1 mmol/L sodium orthovanadate, 50 mmol/L NaF, and 200 μg/L okadaic acid. The lysates were centrifuged for 15 minutes at 12,000 rpm and 4°C. Aliquots of the supernatants (10 μg protein per lane) were separated via 10% or 12% SDS-PAGE and transferred to polyvinylidene difluoride membrane (Immobilon-P; Millipore). Protein was quantified using the BCA Protein Assay Kit (Pierce Chemical Co., Thermo Fisher Scientific, Inc., Rockford, IL). After incubation with primary and horseradish peroxidase–labeled secondary antibody, the complexes were visualized using SuperSignal West Pico Chemiluminescent Substrate (Pierce Chemical Co.) according to the manufacturer’s protocol. For subcellular fractionation, differentiated podocytes were harvested using trypsin-EDTA, and the various cell fractions were isolated using the Subcellular Protein Fractionation Kit (Pierce Chemical Co.) according to the protocol recommended by the manufacturer. The fractions of soluble nuclear extract and chromatin-bound nuclear extract were pooled to achieve a generic nuclear fraction. Plasma membranes were separated using the Membrane Protein Extraction Kit (PromoCell GmbH, Heidelberg, Germany) according to the protocol recommended by the manufacturer. The plasma membrane fraction was dissolved in 0.5% Triton X-100 in PBS. Protein was quantified using Precision Red Protein Assay Reagent (Cytoskeleton) and 10 μg of each preparation separated via 10% SDS-PAGE. Cell size measurements and quantification of focal adhesions were performed using ImageJ software (version 1.44p), as described by Marg et al.28Marg S. Winkler U. Sestu M. Himmel M. Schönherr M. Bär J. Mann A. Moser M. Mierke C.T. Rottner K. Blessing M. Hirrlinger J. Ziegler W.H. The vinculin-DeltaIn20/21 mouse: characteristics of a constitutive, actin-binding deficient splice variant of vinculin.PLoS One. 2010; 5: e11530Crossref PubMed Scopus (21) Google Scholar On the basis of anti-vinculin stainings, grayscale 16-bit images were obtained using a Leica DMLB microscope (×40 magnification) and processed using ImageJ software. With the use of several commands, the program defines the cell edge and measures the cell area. By using the “thresholding” tool to define the contacts and with the command “Subtract background,” the program creates a binary footprint of the adhesion sites. To measure the number of focal adhesions all particles >0.1 μm2 were counted using the “Analyze particles” command. For activity measurements of the small GTPases, we used Rac1, Rho, and Cdc42 Activation Assay Biochem Kits (Cytoskeleton). For each assay, 700 μg protein lysate was incubated with PAK-PBD protein beads (Rac1 and Cdc42) or Rhotekin-RBD beads (RhoA) for 1 hour at 4°C. After washing, bound proteins were separated via 12% SDS-PAGE and blotted on polyvinylidine difluoride membranes. After incubation with antibodies specific for Rac1, RhoA, and Cdc42 and the appropriate secondary antibodies, membranes were developed using SuperSignal West Pico Chemiluminescent Substrate (Pierce Chemical Co.) according to the manufacturer’s protocol. For determination of total amounts of RhoA, Rac1, and Cdc42, 50 μg total protein lysate was used for Western blotting. Total RNA was prepared using the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) following the protocol recommended by the manufacturer with an additional step of DNase digestion (RNase-Free DNase Set; Qiagen). Total RNA, 1 μg, was reverse transcribed using Oligo(dT)15 and random primers and Moloney Murine Leukemia Virus Reverse Transcriptase (Promega GmbH, Mannheim, Germany). Real-time PCR was performed using a Light Cycler 480 (Roche Diagnostics). cDNA was amplified using Fast Start Taq Polymerase (Roche Diagnostics), SYBR Green (Invitrogen), gene-specific primers, and the following PCR conditions: 5 minutes at 95°C for 45 cycles, and 10 seconds at 95°C, 10 seconds at 60°C, and 10 seconds at 72°C. Specificity of the amplification product was verified via melting curve analysis. The samples were measured as multiplexed reactions and normalized to the constitutive gene mouse hypoxanthine phosphoribosyltransferase 1. All primers for the listed transcripts were designed using Primer3 software: PKCι: 5′-AGGAACGATTGGGTTGTCAC-3′, 5′-GGCAAGCAGAATCAGACACA-3′; PKCζ: 5′-GCCTCCCTTCCAGCCCCAGA-3′, 5′-CACGGACTCCTCAGCAGACAGCA-3′; Def-6: 5′-CACCAACGTGAAACACTGGA-3′, 5′-TGGTGGTGGGTCGCTTAT-3′; and HPRT-1: 5′-CAGTCCCAGCGTCGTGATTA-3′, 5′-AGCAAGTCTTTCAGTCCTGTC-3′ The Whole Mouse Genome Oligo Microarray (G4122F, ID 014868; Agilent Technologies, Inc., Santa Clara, CA) used in the present study contains 45,018 oligonucleotide probes covering the entire murine transcriptome. Synthesis of Cy3-labeled cRNA was performed using the Quick Amp Labeling kit, one color (No. 5190-0442; Agilent Technologies) according to the manufacturer’s recommendations. cRNA fragmentation, hybridization, and washing steps were also performed exactly as recommended, using the One-color Microarray-Based Gene Expression Analysis Protocol version 5.7. Slides were scanned using the Agilent Micro Array Scanner G2565CA at two different photomultiplier tube settings (100% and 5%) to increase the dynamic range of the measurements (extended dynamic range mode). Data extraction was performed using Feature Extraction Software version 10.7.1.1 by using the recommended default extraction protocol file: GE1_107_sep09.xml. Processed intensity values of the green channel (gProcessedSignal, or gPS) were normalized via global linear scaling: All gPS values of one sample were multiplied by an array-specific factor. This scaling factor was calculated by dividing a reference 75th percentile value (set as 1500 for the entire series) by the 75th percentile value of the particular microarray (Array i in the formula below). Accordingly, normalized gPS values for all samples (microarray data sets) were calculated using the following formula: Normalized gPSArray i = gPSArray i × (75th PercentileReference Array/75th PercentileArray i). A lower intensity threshold was defined as 1% of the reference 75th percentile value (threshold = 15). All of those normalized gPS values that fell below this intensity border were substituted for by the respective surrogate value of 15. Calculation of ratio values of relative gene expression was performed using Excel macros. Heat maps were generated using the MultiExperiment Viewer 4.5.1 program. The intensity values of all genes were logarithmized, and the mean intensity value of each single gene was subtracted from all values. These data were loaded into the MultiExperiment Viewer and clustered using the command “Hierarchical clustering” and the adjustments “Euclidean distance” and “Average linkage clustering.” For quantification of Def-6 expression, cryosections of wild-type (WT) and knockout kidneys were double stained with nidogen and Def-6. Def-6 expression in the podocytic area of glomeruli was rated using a semiquantitative score (0 = none, 1 = less, 2 = median, 3 = enhanced, 4 = strong) by a scientist blinded to the study (M.S.). From each genotype, 40 glomeruli from four animals were scored. The podocytic area was determined by subtraction of the mesangial area marked by nidogen and the Def-6–positive area in the glomeruli. Data are given as means ± SD and were compared using unpaired Student's t-tests. Data analysis was performed using Excel statistical software. Significant differences were accepted at P < 0.05. In a previous study, we identified PKCλ/ι as a critical factor for podocyte polarity and architecture.5Huber T.B. Hartleben B. Winkelmann K. Schneider L. Becker J.U. Leitges M. Walz G. Haller H. Schiffer M. Loss of podocyte aPKClambda/iota causes polarity defects and nephrotic syndrome.J Am Soc Nephrol. 2009; 20: 798-806Crossref PubMed Scopus (81) Google Scholar To generate a PKCλ/ι-deficient podocyte cell line, we crossed PKCλ/ι flox/flox Podocin-Cre mice with Immorto transgenic animals expressing the temperature-sensitive SV40 large T antigen (tsA58TAg) under control of the γ-interferon–inducible H-2Kb promoter.25Jat P.S. Noble M.D. Ataliotis P. Tanaka Y. Yannoutsos N. Larsen L. Kioussis D. Direct derivation of conditionally immortal cell lines from an H-2Kb-tsA58 transgenic mouse.Proc Natl Acad Sci U S A. 1991; 88: 5096-5100Crossref PubMed Scopus (628) Google Scholar Glomeruli were extracted from deficient and control mice, outgrowing cells were isolated, and cell lines were cultured from single cells. To compare the two atypical PKC isoforms PKCλ/ι and PKCζ in cell culture experiments, we also generated monoclonal cell lines from mice deficient in PKCζ6Leitges M. Sanz L. Martin P. Duran A. Braun U. Garcia J.F. Camacho F. Diaz-Meco M.T. Rennert P.D. Moscat J. Targeted disruption of the zetaPKC gene results in the impairment of the NF-kappaB pathway.Mol Cell. 2001; 8: 771-780Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar and control mice of the same strain. Three cell lines of each genotype were characterized using PCR analysis and various podocyte-specific antibodies (Supplemental Figure S1). After differentiation, podocyte marker proteins synaptopodin29Mundel P. Gilbert P. Kriz W. Podocytes in glomerulus of rat kidney express a characteristic 44 KD protein.J Histochem Cytochem. 1991; 39: 1047-1056Crossref PubMed Scopus (129) Google Scholar and WT-130Mundlos S. Pelletier J. Darveau A. Bachmann M. Winterpacht A. Zabel B. Nuclear localization of the protein encoded by the Wilms’ tumor gene WT1 in embryonic and adult tissues.Development. 1993; 119: 1329-1341PubMed Google Scholar were detected in all cell lines via immunofluorescence staining, and expression of nephrin, podocin, and WT-1 were confirmed in cell lysates via Western blot analysis (data not shown). Under permissive conditi" @default.
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• W2031389630 date "2013-12-01" @default.
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• W2031389630 title "Def-6, a Novel Regulator of Small GTPases in Podocytes, Acts Downstream of Atypical Protein Kinase C (aPKC) λ/ι" @default.
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