Construction of a viral T2A-peptide based knock-in mouse model for enhanced Cre recombinase activity and fluorescent labeling of podocytes
2017; Elsevier BV; Volume: 91; Issue: 6 Linguagem: Inglês
10.1016/j.kint.2016.12.011
ISSN1523-1755
AutoresSybille Koehler, Sebastian Brähler, Fabian Braun, Henning Hagmann, Markus M. Rinschen, Martin R. Späth, Martin Höhne, F. Thomas Wunderlich, Bernhard Schermer, Thomas Benzing, Paul T. Brinkkoetter,
Tópico(s)Systemic Lupus Erythematosus Research
ResumoPodocyte injury is a key event in glomerular disease leading to proteinuria and opening the path toward glomerular scarring. As a consequence, glomerular research strives to discover molecular mechanisms and signaling pathways affecting podocyte health. The hNphs2.Cre mouse model has been a valuable tool to manipulate podocyte-specific genes and to label podocytes for lineage tracing and purification. Here we designed a novel podocyte-specific tricistronic Cre mouse model combining codon improved Cre expression and fluorescent cell labeling with mTomato under the control of the endogenous Nphs2 promoter using viral T2A-peptides. Independent expression of endogenous podocin, codon improved Cre, and mTomato was confirmed by immunofluorescence, fluorescent activated cell sorting and protein analyses. Nphs2pod.T2A.ciCre.T2A.mTomato/wild-type mice developed normally and did not show any signs of glomerular disease or off-target effects under basal conditions and in states of disease. Nphs2pod.T2A.ciCre.T2A.mTomato/wild-type-mediated gene recombination was superior to conventional hNphs2.Cre mice–mediated gene recombination. Last, we compared Cre efficiency in a disease model by mating Nphs2pod.T2A.ciCre.T2A.mTomato/wild-type and hNphs2.Cre mice to Phb2fl/fl mice. The podocyte-specific Phb2 knockout by Nphs2pod.T2A.ciCre.T2A.mTomato/wild-type mice resulted in an aggravated glomerular injury as compared to a podocyte-specific Phb2 gene deletion triggered by hNphs2.Cre. Thus, we generated the first tricistronic podocyte mouse model combining enhanced Cre recombinase efficiency and fluorescent labeling in podocytes without the need for additional matings with conventional reporter mouse lines. Podocyte injury is a key event in glomerular disease leading to proteinuria and opening the path toward glomerular scarring. As a consequence, glomerular research strives to discover molecular mechanisms and signaling pathways affecting podocyte health. The hNphs2.Cre mouse model has been a valuable tool to manipulate podocyte-specific genes and to label podocytes for lineage tracing and purification. Here we designed a novel podocyte-specific tricistronic Cre mouse model combining codon improved Cre expression and fluorescent cell labeling with mTomato under the control of the endogenous Nphs2 promoter using viral T2A-peptides. Independent expression of endogenous podocin, codon improved Cre, and mTomato was confirmed by immunofluorescence, fluorescent activated cell sorting and protein analyses. Nphs2pod.T2A.ciCre.T2A.mTomato/wild-type mice developed normally and did not show any signs of glomerular disease or off-target effects under basal conditions and in states of disease. Nphs2pod.T2A.ciCre.T2A.mTomato/wild-type-mediated gene recombination was superior to conventional hNphs2.Cre mice–mediated gene recombination. Last, we compared Cre efficiency in a disease model by mating Nphs2pod.T2A.ciCre.T2A.mTomato/wild-type and hNphs2.Cre mice to Phb2fl/fl mice. The podocyte-specific Phb2 knockout by Nphs2pod.T2A.ciCre.T2A.mTomato/wild-type mice resulted in an aggravated glomerular injury as compared to a podocyte-specific Phb2 gene deletion triggered by hNphs2.Cre. Thus, we generated the first tricistronic podocyte mouse model combining enhanced Cre recombinase efficiency and fluorescent labeling in podocytes without the need for additional matings with conventional reporter mouse lines. The kidney filtration barrier consists of fenestrated endothelial cells, the glomerular basement membrane, and highly specialized epithelial cells, the podocytes.1Pavenstädt H. Kriz W. Kretzler M. Cell biology of the glomerular podocyte.Physiol Rev. 2003; 83: 253-307Crossref PubMed Scopus (1203) Google Scholar Podocyte injury and the development of albuminuria are hallmarks of glomerular disease and are considered to be the common fate-determining path.2Mundel P. Shankland S.J. Podocyte biology and response to injury.J Am Soc Nephrol. 2002; 13: 3005-3015Crossref PubMed Scopus (580) Google Scholar, 3Schiffer M. Teng B. Gu C. et al.Pharmacological targeting of actin-dependent dynamin oligomerization ameliorates chronic kidney disease in diverse animal models.Nat Med. 2015; 21: 601-609Crossref PubMed Scopus (89) Google Scholar, 4Brinkkoetter P.T. Ising C. Benzing T. The role of the podocyte in albumin filtration.Nat Rev Nephrol. 2013; 9: 328-336Crossref PubMed Scopus (160) Google Scholar The hNphs2.Cre (hPod.Cre) mouse has been the backbone of podocyte research despite certain limitations.5Moeller M.J. Sanden S.K. Soofi A. et al.Podocyte-specific expression of cre recombinase in transgenic mice.Genesis. 2003; 35: 39-42Crossref PubMed Scopus (249) Google Scholar Due to the random integration of the hPod.Cre transgene and the prokaryotic nucleotide sequence, Cre expression is limited and might be affected by gene silencing. A yet unmet challenge in podocyte research lies in the lack of in vitro models that mimic the podocyte's in vivo environment. Isolated primary podocytes are considered to be the best approximation. However, culturing primary podocytes is technically demanding. For efficient purification of primary podocytes, additional breedings with a reporter line are required. These are time-consuming and cost intensive. We set out to generate a podocyte-specific Cre recombinase mouse line that allows for improved Cre expression and simultaneous fluorescent labeling in podocytes. Here, we describe a tricistronic mouse model combining expression of a codon-improved Cre recombinase6Shimshek D.R. Kim J. Hübner M.R. et al.Codon-improved Cre recombinase (iCre) expression in the mouse.Genesis. 2002; 32: 19-26Crossref PubMed Scopus (290) Google Scholar with expression of a membrane-targeted tandem dimer tdTomato (mTomato) under the control of the endogenous Nphs2 promoter.7Shaner N.C. Campbell R.E. Steinbach P.A. et al.Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.Nat Biotechnol. 2004; 22: 1567-1572Crossref PubMed Scopus (3496) Google Scholar The widely used Cre recombinase originates from the bacteriophage P1.6Shimshek D.R. Kim J. Hübner M.R. et al.Codon-improved Cre recombinase (iCre) expression in the mouse.Genesis. 2002; 32: 19-26Crossref PubMed Scopus (290) Google Scholar Shimshek et al.6Shimshek D.R. Kim J. Hübner M.R. et al.Codon-improved Cre recombinase (iCre) expression in the mouse.Genesis. 2002; 32: 19-26Crossref PubMed Scopus (290) Google Scholar introduced a codon-improved nucleotide sequence (ciCre) adapted to eukaryotic species by minimizing the CpG (5′—C—phosphate—G—3′) content to reduce the chances of epigenetic gene silencing and altering the stop codon leading to increased Cre expression. To link ciCre expression to a fluorescent reporter and to ascertain exclusive expression in podocytes, we used the 2A-peptide approach and integrated the sequences into the endogenous Nphs2 locus by gene targeting. The 2A-peptides are 19 amino acid–long viral sequences that induce a discontinuity in the translation process at glycine 18 and proline 19.8de Felipe P. Luke G.A. Brown J.D. et al.Inhibition of 2A-mediated "cleavage" of certain artificial polyproteins bearing N-terminal signal sequences.Biotechnol J. 2010; 5: 213-223Crossref PubMed Scopus (64) Google Scholar, 9Doronina V.A. Wu C. de Felipe P. et al.Site-specific release of nascent chains from ribosomes at a sense codon.Mol Cell Biol. 2008; 28: 4227-4239Crossref PubMed Scopus (124) Google Scholar As a consequence, the first peptide is released from the ribosome with a C-terminal 2A-tag.9Doronina V.A. Wu C. de Felipe P. et al.Site-specific release of nascent chains from ribosomes at a sense codon.Mol Cell Biol. 2008; 28: 4227-4239Crossref PubMed Scopus (124) Google Scholar At proline 19, the translation process starts again and a second downstream product is expressed.9Doronina V.A. Wu C. de Felipe P. et al.Site-specific release of nascent chains from ribosomes at a sense codon.Mol Cell Biol. 2008; 28: 4227-4239Crossref PubMed Scopus (124) Google Scholar Hence, 2A-peptides lead to the equimolar coexpression of different proteins from a single open reading frame under the control of a single promoter.8de Felipe P. Luke G.A. Brown J.D. et al.Inhibition of 2A-mediated "cleavage" of certain artificial polyproteins bearing N-terminal signal sequences.Biotechnol J. 2010; 5: 213-223Crossref PubMed Scopus (64) Google Scholar, 9Doronina V.A. Wu C. de Felipe P. et al.Site-specific release of nascent chains from ribosomes at a sense codon.Mol Cell Biol. 2008; 28: 4227-4239Crossref PubMed Scopus (124) Google Scholar, 10Kim J.H. Lee S.R. Li L.H. et al.High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice.PLoS One. 2011; 6: e18556Crossref PubMed Scopus (844) Google Scholar, 11Chng J. Wang T. Nian R. et al.Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells.MAbs. 2015; 7: 403-412Crossref PubMed Scopus (85) Google Scholar To achieve equimolar expression of ciCre, mTomato, and podocin, we linked the endogenous Nphs2 with the ciCre and mTomato sequences by viral Thosea asigna virus 2A (T2A) sequences (Figure 1a and b).12Toyoda H. Nicklin M.J. Murray M.G. et al.A second virus-encoded proteinase involved in proteolytic processing of poliovirus polyprotein.Cell. 1986; 45: 761-770Abstract Full Text PDF PubMed Scopus (254) Google Scholar, 13Donnelly M.L. Luke G. Mehrotra A. et al.Analysis of the aphthovirus 2A/2B polyprotein "cleavage" mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal "skip".J Gen Virol. 2001; 82: 1013-1025Crossref PubMed Scopus (556) Google Scholar The T2A.ciCre.T2A.mTomato sequence was inserted between the last amino acid of exon 8 of Nphs2 and the stop codon (Figure 1a). Southern blots confirm correct homologous recombination at the 3′ and 5′ side as well as single integration (Figure 1c). Expression of ciCre and mTomato were evaluated by immunofluorescence microscopy (Figure 1d and e). Both ciCre and mTomato exhibited a podocyte-specific expression pattern with a nuclear localization of ciCre and a membrane-associated pattern of mTomato (Figure 1d and e). Based on mTomato expression, we were able to isolate primary podocytes of Nphs2pod.T2A.ciCre.T2A.mTomato/wild-type (mPod.2A.ciCre) mice by fluorescent activated cell sorting (Figure 2a). Detection of the T2A sequence by Western blotting revealed 2 distinct bands (50 kDa podocin.T2A, 43 kDa ciCre.T2A), proving that the translation discontinuity due to the viral T2A sequence is functional (Figure 2b). Podocin expression levels in glomerular lysates were not altered despite the fact that we observed a double band as the T2A-peptide remains at the C-terminus (Figure 2c and d). In addition, we did not observe any differences in the total glomerular proteome or with respect to the expression of podocyte-specific proteins (Figure 2d and e).Figure 2Validation of independent podocin, codon-improved Cre (ciCre), and mTomato expression. (a) Fluorescent activated cell (FAC) sorting of primary glomerular cells showed a clear peak representing mTomato-positive cells and therefore the possibility of using FAC sorting. (b) Staining of glomerular lysates with an anti-Thosea asigna virus 2A (anti-T2A) antibody showed single bands for podocin.T2A (50 kDa) and ciCre.T2A (43 kDa), proving independent expression of the proteins. A fusion product at 145 kDa could not be observed. (c) Western blot analysis of glomerular lysates from wild-type (WT) and (mPod.2A.ciCre) mice showed similar expression levels of podocin. Lysates from mPod.2A.ciCre mice showed a double band as a result of the heterozygous expression of the podocin (WT) and podocin.T2A protein. Quantification of 3 independent experiments analyzing the double and single band by densitometry revealed no significant difference in total podocin amount comparing WT and mPod.2A.ciCre lysates (mean ± SEM, paired t test: NS: P > 0.05). (d) Label-free proteomic analysis of the glomerular proteome from mPod.2A.ciCre and WT mice. Glomeruli were lysed in urea and subjected to quantitative proteomic analysis using n–liquid chromatography–mass spectrometry/mass spectrometry (nano–LC-MS/MS). The volcano plot plots log2-fold change between mPod.2A.ciCre and WT mice versus the significance of the change. Proteins beyond the line represent significantly changed protein (false discovery rate [FDR] 0.2, s0 = 1). Comparison of the glomerular proteome from WT and mPod.2A.ciCre mice did not reveal significant differences in protein expression. Proteins important for podocyte homeostasis, such as nephrin (Nphs1), podocin (Nphs2), Cd2ap (Cd2ap), synaptopodin (Synpo), as well as house-keeping genes (Gapdh, Actb) are marked with their respective gene symbols. (e) Analyses of the normalized label free quantification (LFQ) of nephrin, podocin, CD2AP, synaptopodin, and NEPH1 did not reveal significant differences in expression levels comparing mPod.2A.ciCre and WT mice (mean ± SEM, 2-way analysis of variance with Sidak multiple comparisons test: NS: P > 0.05).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To exclude a disease-causing effect as a consequence of the podocin modifications at the C-terminus or coexpression of ciCre and mTomato, we monitored mPod.2A.ciCre mice for 12 months. The mPod.2A.ciCre mice developed normally and showed normal renal function (Figure 3a–f). To address whether the C-terminal podocin modification may cause subtle changes and render the podocytes susceptible for disease, we challenged mPod.2A.ciCre mice with lipopolysaccharide (LPS). Again, we did not observe any differences in the urinary albumin-to-creatinine ratios between mPod.2A.ciCre and wild-type (WT) mice (Figure 3g). To verify that Cre recombinase expression under the control of the endogenous Nphs2 promoter and the more efficient translation due to the improved codon usage leads to higher Cre expression,6Shimshek D.R. Kim J. Hübner M.R. et al.Codon-improved Cre recombinase (iCre) expression in the mouse.Genesis. 2002; 32: 19-26Crossref PubMed Scopus (290) Google Scholar we mated ROSA26mTmG/WT (mTmG: membrane-targeted tandem dimer Tomato/membrane-targeted green fluorescent protein) reporter mice with mPod.2A.ciCre or hPod.Cre14Muzumdar M.D. Tasic B. Miyamichi K. et al.A global double-fluorescent Cre reporter mouse.Genesis. 2007; 45: 593-605Crossref PubMed Scopus (2265) Google Scholar mice and analyzed green fluorescent protein and Cre expression by Western blotting (Figure 4a). The mPod.2A.ciCre;mTmG mice showed increased Cre (1.435-fold increase) and green fluorescent protein expression (1.809-fold increase) as compared to hPod.Cre;mTmG mice (Figure 4a). These findings are in line with previous results by Shimshek et al.6Shimshek D.R. Kim J. Hübner M.R. et al.Codon-improved Cre recombinase (iCre) expression in the mouse.Genesis. 2002; 32: 19-26Crossref PubMed Scopus (290) Google Scholar who reported a 1.8-fold increase of recombination efficiency. Last, we tested the mPod.2A.ciCre mouse in a published genetic model of focal segmental glomerulosclerosis.15Ising C. Koehler S. Brähler S. et al.Inhibition of insulin/IGF-1 receptor signaling protects from mitochondria-mediated kidney failure.EMBO Mol Med. 2015; 7: 275-287Crossref PubMed Scopus (53) Google Scholar We mated mPod.2A.ciCre mice with Phb2fl/fl mice to generate a podocyte-specific loss of Phb2. As published, loss of Phb2 in podocytes led to an early onset of albuminuria, glomerulosclerosis, and premature death.15Ising C. Koehler S. Brähler S. et al.Inhibition of insulin/IGF-1 receptor signaling protects from mitochondria-mediated kidney failure.EMBO Mol Med. 2015; 7: 275-287Crossref PubMed Scopus (53) Google Scholar Surprisingly, the mean survival of mPod.2A.ciCre;Phb2fl/fl mice was 16 days shorter than that of hPod.Cre;Phb2fl/fl mice (Figure 4b). Although considerable morphological changes in mPod.2A.ciCre;Phb2fl/fl mice were observed (Figure 4c and d), protein expression of mTomato was relatively stable during the course of the glomerular disease (Figure 4d). We generated a novel mouse model for podocyte-specific gene manipulation and simultaneous fluorescent labeling of podocytes. To this end, we targeted exon 8 of the Nphs2 gene and integrated a T2A.ciCre.T2A.mTomato sequence between the last amino acid of podocin and the stop codon. The targeted insertion of ciCre is expected to be less prone to creating epigenetic off-target effects and to lead to increased Cre recombinase expression levels.6Shimshek D.R. Kim J. Hübner M.R. et al.Codon-improved Cre recombinase (iCre) expression in the mouse.Genesis. 2002; 32: 19-26Crossref PubMed Scopus (290) Google Scholar By targeting the endogenous Nphs2 locus, we expect not only to target literally all podocytes but also to do so in a cell-specific manner. This tricistronic podocyte-specific mouse combines exquisite cell-type specificity with all the advantages of a codon-improved Cre recombinase driver line with fluorescent membrane-targeted labeling without the need for additional time- and cost-intensive animal matings. The 2A approach allows the generation of transgenic mouse lines with stoichiometric coexpression of at least up to 4 different proteins of interest including different fluorescence markers.16Roulston C. Luke G.A. de Felipe P. et al."2A-like" signal sequences mediating translational recoding: a novel form of dual protein targeting.Traffic. 2016; 17: 923-939Crossref PubMed Scopus (15) Google Scholar, 17Minskaia E. Nicholson J. Ryan M.D. Optimisation of the foot-and-mouth disease virus 2A co-expression system for biomedical applications.BMC Biotechnol. 2013; 13: 67Crossref PubMed Scopus (48) Google Scholar, 18Szymczak A.L. Workman C.J. Wang Y. et al.Correction of multi-gene deficiency in vivo using a single "self-cleaving" 2A peptide–based retroviral vector.Nat Biotechnol. 2004; 22: 589-594Crossref PubMed Scopus (918) Google Scholar The 2A technology circumvents the disadvantages of conventional coexpression approaches such as promoter interference when multiple promoters have been employed, steric effects of fusion proteins that often lead to loss of function of the fused proteins, or internal ribosomal entry site elements with increased nucleotide length, and reduced expression levels of the proteins downstream of the internal ribosomal entry site sequence.8de Felipe P. Luke G.A. Brown J.D. et al.Inhibition of 2A-mediated "cleavage" of certain artificial polyproteins bearing N-terminal signal sequences.Biotechnol J. 2010; 5: 213-223Crossref PubMed Scopus (64) Google Scholar, 19Pfützner W. Retroviral bicistronic vectors.Drug News Perspect. 2008; 21: 473-480Crossref PubMed Scopus (12) Google Scholar, 20Martínez-Salas E. Internal ribosome entry site biology and its use in expression vectors.Curr Opin Biotechnol. 1999; 10: 458-464Crossref PubMed Scopus (164) Google Scholar, 21Wong E.T. Ngoi S.M. Lee C.G. Improved co-expression of multiple genes in vectors containing internal ribosome entry sites (IRESes) from human genes.Gene Ther. 2002; 9: 337-344Crossref PubMed Scopus (57) Google Scholar, 22Kozak M. A second look at cellular mRNA sequences said to function as internal ribosome entry sites.Nucleic Acids Res. 2005; 33: 6593-6602Crossref PubMed Scopus (127) Google Scholar, 23Baranick B.T. Lemp N.A. Nagashima J. et al.Splicing mediates the activity of four putative cellular internal ribosome entry sites.Proc Natl Acad Sci U S A. 2008; 105: 4733-4738Crossref PubMed Scopus (63) Google Scholar The remaining 2A-peptide at the C-terminus represents a specific tag for which antibodies are available.8de Felipe P. Luke G.A. Brown J.D. et al.Inhibition of 2A-mediated "cleavage" of certain artificial polyproteins bearing N-terminal signal sequences.Biotechnol J. 2010; 5: 213-223Crossref PubMed Scopus (64) Google Scholar, 9Doronina V.A. Wu C. de Felipe P. et al.Site-specific release of nascent chains from ribosomes at a sense codon.Mol Cell Biol. 2008; 28: 4227-4239Crossref PubMed Scopus (124) Google Scholar, 11Chng J. Wang T. Nian R. et al.Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells.MAbs. 2015; 7: 403-412Crossref PubMed Scopus (85) Google Scholar There are certain limitations associated with this technique that need to be taken into account. The insertion of the T2A.ciCre.T2A.mTomato sequence results in a tagged podocin and this modification could change expression levels or posttranslational modifications or alter signaling events. To uncover overt limitations of this mouse model, we performed functional and proteomics studies. Functionally, we did not observe any glomerular pathology during a 12-month study period as well as no significant differences in a disease model (Figure 3). We performed quantitative analyses of the glomerular proteome using label-free quantitative proteomics. There were no obvious differences in the global glomerular proteome or the expression levels of essential podocyte proteins. Although this method does not address abundant glomerular posttranslational modifications,24Rinschen M.M. Wu X. König T. et al.Phosphoproteomic analysis reveals regulatory mechanisms at the kidney filtration barrier.J Am Soc Nephrol. 2014; 25: 1509-1522Crossref PubMed Scopus (36) Google Scholar this information argues against a global, significant detrimental effect of the Pod.2A.Cre protein. In addition, higher expression levels of Cre recombinase could cause off-target effects that might bias experimental studies. However, the codon-improved Cre recombinase sequence was established more than a decade ago and since then has been successfully applied in various tissues.6Shimshek D.R. Kim J. Hübner M.R. et al.Codon-improved Cre recombinase (iCre) expression in the mouse.Genesis. 2002; 32: 19-26Crossref PubMed Scopus (290) Google Scholar Here, we provide experimental evidence that the observed aggravated phenotype in the Phb2-model is the result of increased specific Cre expression. In conclusion, we established a tricistronic podocyte mouse model that combines highly specific enhanced Cre recombinase activity with fluorescent labeling. This mouse model represents a novel genetic tool that will significantly facilitate podocyte research in the future. Nphs2.T2A.ciCre.T2A.mTomato mice were generated by Taconic Biosciences (Cologne, Germany). The targeting vector has been generated using bacterial artificial chromosome clones from the C57BL/6J RPCIB-731 BAC library and was transfected into the TaconicArtemis C57BL/6N Tac embryonic stem cell line. Homologous recombinant clones were isolated using positive (PuroR) and negative (Thymidine kinase) selection. Southern blot analyses were performed to verify correct homologous recombination at the 3′ and 5′ sides as well as single integration. The constitutive knock-in allele was obtained after in vivo Flp-mediated removal of the selection marker. A glycine-serine-glycine linker was integrated between the upstream protein and the T2A-peptide to achieve highest cleavage efficiency.11Chng J. Wang T. Nian R. et al.Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells.MAbs. 2015; 7: 403-412Crossref PubMed Scopus (85) Google Scholar Basal studies and the LPS administration were performed with heterozygous animals. To validate Cre efficiency, (mPod.2A.ciCre) mice were mated with R26mTmG mice and compared with hPod.Cretg/WT; R26mTmG mice.14Muzumdar M.D. Tasic B. Miyamichi K. et al.A global double-fluorescent Cre reporter mouse.Genesis. 2007; 45: 593-605Crossref PubMed Scopus (2265) Google Scholar Isolation of glomeruli, preparation of a glomerular single-cell suspension and fluorescent activated cell sorting was done as previously described.25Wanner N. Hartleben B. Herbach N. et al.Unraveling the role of podocyte turnover in glomerular aging and injury.J Am Soc Nephrol. 2014; 25: 707-716Crossref PubMed Scopus (141) Google Scholar To generate a podocyte-specific Phb2 knockout, (mPod.2A.ciCre) mice were mated to Phb2fl/fl mice.15Ising C. Koehler S. Brähler S. et al.Inhibition of insulin/IGF-1 receptor signaling protects from mitochondria-mediated kidney failure.EMBO Mol Med. 2015; 7: 275-287Crossref PubMed Scopus (53) Google Scholar For comparison, Phb2fl/fl mice were also mated to (hPod.Cre) mice.5Moeller M.J. Sanden S.K. Soofi A. et al.Podocyte-specific expression of cre recombinase in transgenic mice.Genesis. 2003; 35: 39-42Crossref PubMed Scopus (249) Google Scholar, 15Ising C. Koehler S. Brähler S. et al.Inhibition of insulin/IGF-1 receptor signaling protects from mitochondria-mediated kidney failure.EMBO Mol Med. 2015; 7: 275-287Crossref PubMed Scopus (53) Google Scholar For all studies, both sexes were used and all animals were on a pure C57/Bl6 background. Urine was collected at the time points mentioned. Blood was taken after anesthesia from the heart. Mouse holding was done in the University of Cologne animal facility according to standardized specific pathogen-free conditions. The experimental protocol was examined and approved by the Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen (State Agency for Nature, Environment, and Consumer Protection North Rhine-Westphalia). Whole kidneys from mPod.2A.ciCre;ROSA26mTmG/WT and hPod.Cre;ROSA26mTmG/WT mice were homogenized with a glass homogenizer and lysed with a modified radio immunoprecipitation assay buffer (1% Igepal CA-630 [Sigma, St. Louis, MO], 150 mmol/l NaCl, 0.25% sodium deoxycholate, and 50 mmol/l tris[hydroxymethyl]-aminomethane) with a protease inhibitor mix. Lysates were used for sodium dodecylsulfate–polyacrylamide gel electrophoresis. Glomeruli were isolated as mentioned before and lysed with a buffer containing 2% sodium dodecylsulfate in 50 mmol/l tris(hydroxymethyl)-aminomethane at pH 8.0. Denaturated lysates (whole kidney and glomeruli) were used for sodium dodecylsulfate–polyacrylamide gel electrophoresis, followed by Western blotting of the proteins on a polyvinylidene diflouride membrane. Membranes were incubated with the respective primary (see Table 1) and secondary antibodies and signals were visualized using enhanced chemiluminescence.Table 1AntibodiesNameCompanyCatalog no.Host speciesDilution IFDilution WBAnti-2AMilliporeABS31rabbit1:500Anti-CreNovagen69050-3rabbit1:1001:500Anti-GFPSanta Cruzsc-9996mouse1:500Anti-podocinSigmaP0372rabbit1:100Anti-tubulinSigmamouse1:1000GFP, green fluorescent protein; IF, immunofluorescence; WB, Western blot. Open table in a new tab GFP, green fluorescent protein; IF, immunofluorescence; WB, Western blot. Glomeruli were lysed in urea, and proteins were reduced with dithiothreitol, alkylated with iodoacetamide in the dark, and digested with trypsin 1:100 weight-weight ratio as previously described.24Rinschen M.M. Wu X. König T. et al.Phosphoproteomic analysis reveals regulatory mechanisms at the kidney filtration barrier.J Am Soc Nephrol. 2014; 25: 1509-1522Crossref PubMed Scopus (36) Google Scholar Peptides were cleaned using stage tips as previously described.26Rappsilber J. Mann M. Ishihama Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips.Nat Protoc. 2007; 2: 1896-1906Crossref PubMed Scopus (2588) Google Scholar Quantitative label-free proteomic analysis was performed on a QExactive Plus coupled to n–liquid chromatography as described previously. Raw files were searched against a recent mouse uniprot database (downloaded February 24, 2015) using MaxQuant version 1.5.0.1 (MPI of Biochemistry, Martinsried, Germany) using default settings and with the label-free quantification option enabled.27Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9210) Google Scholar Txt file output containing expression levels of was filtered (reverse and contaminant proteins were removed) and normalized in Perseus by subtraction of the median (version 1.5.0.1) as previously described.28Rinschen M.M. Bharill P. Wu X. et al.The ubiquitin ligase Ubr4 controls stability of podocin/MEC-2 supercomplexes.Hum Mol Genet. 2016; 25: 1328-1344Crossref PubMed Scopus (39) Google Scholar Perfusion of mice was performed with phosphate-buffered saline via the heart. Renal tissue was either embedded and frozen in Tissue-Tek O.C.T. compound (Sakura, Leiden, the Netherlands) or fixed in 4% formaldehyde. For immunofluorescence analysis, frozen kidneys were cut into 5-μm sections and fixed with 4% formaldehyde for 2 minutes. After blocking with 5% normal donkey serum for 30 minutes, sections were incubated with primary antibody (see Table 1) for 1 hour at room temperature. Following 3 washes with phosphate-buffered saline + 0.1% Triton-X sections were incubated for 45 minutes with a secondary antibody. Mounting was performed with Prolong Gold antifade with 4′,6-diamidino-2-phenylindole (Thermo Fisher Scientific, Waltham, MA). Formaldehyde-fixed renal tissue was embedded in paraffin and cut into 2-μm thick sections. For histologic analysis, periodic-acid staining was performed. All images were acquired with an Axiovert 200 M microscope/C-Apochromat 63X/1.20 water immersion objective (all from Carl Zeiss MicroImaging GmbH, Oberkochen, Germany). All images were further processed with ImageJ (National Institutes of Health, Bethesda, MD)/Fiji software (Madison, WI, USA). To measure the urinary albumin-to-creatinine ratio during aging, a mouse albumin enzyme-linked immunosorbent assay kit (ICL/Dunn Labortechnik GmbH, Asbach, Germany) and a urinary creatinine kit (Biomol, Hamburg, Germany) were used according to the manufacturers' descriptions. LPS was injected i.p. into mPod.2A.ciCre and WT mice at an age of 12 to 15 weeks. After collecting baseline urine, 0.5 μg/g body weight LPS (dissolved in NaCl: 0.2 μg/μl) was injected.29Lorenzen J. Shah R. Biser A. et al.The role of osteopontin in the development of albuminuria.J Am Soc Nephrol. 2008; 19: 884-890Crossref PubMed Scopus (68) Google Scholar Twenty-four hours after the injection, urine was collected and animals were killed. All the authors declared no conflict of interests. We thank M. Bruetting, G. Rappl, and the Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases imaging facility for excellent technical support. This work was supported by scholarships of the German Kidney Foundation (Deutsche Nierenstiftung) and Köln Fortune Program of the University of Cologne, Germany to SB. BS obtained funding from the Deutsche Forschungsgemeinschaft (SCHE1562/2). TB received funding from the Deutsche Forschungsgemeinschaft (SFB635 and 829). PTB received funding from the Deutsche Forschungsgemeinschaft (BR2955/4-1 and BR2955/6-1) and Köln Fortune Program of the University of Cologne, Germany.
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