Innate Immune Signaling Contributes to Tubular Cell Senescence in the Glis2 Knockout Mouse Model of Nephronophthisis
2019; Elsevier BV; Volume: 190; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2019.09.013
ISSN1525-2191
AutoresHeng Jin, Yan Zhang, Dingxiao Liu, Shan Shanshan Wang, Qiong Ding, Prerna Rastogi, Madison Purvis, Angela Yee‐Moon Wang, Sarah El-Hadi, Chongyu Ren, Chao Cao, Yanfen Chai, Peter Igarashi, Anton M. Jetten, Dongmei Lu, Massimo Attanasio,
Tópico(s)Telomeres, Telomerase, and Senescence
ResumoNephronophthisis (NPHP), the leading genetic cause of end-stage renal failure in children and young adults, is a group of autosomal recessive diseases characterized by kidney-cyst degeneration and fibrosis for which no therapy is currently available. To date, mutations in >25 genes have been identified as causes of this disease that, in several cases, result in chronic DNA damage in kidney tubular cells. Among such mutations, those in the transcription factor–encoding GLIS2 cause NPHP type 7. Loss of function of mouse Glis2 causes senescence of kidney tubular cells. Senescent cells secrete proinflammatory molecules that induce progressive organ damage through several pathways, among which NF-κB signaling is prevalent. Herein, we show that the NF-κB signaling is active in Glis2 knockout kidney epithelial cells and that genetic inactivation of the toll-like receptor (TLR)/IL-1 receptor or pharmacologic elimination of senescent cells (senolytic therapy) reduces tubule damage, fibrosis, and apoptosis in the Glis2 mouse model of NPHP. Notably, in Glis2, Tlr2 double knockouts, senescence was also reduced and proliferation was increased, suggesting that loss of TLR2 activity improves the regenerative potential of tubular cells in Glis2 knockout kidneys. Our results further suggest that a combination of TLR/IL-1 receptor inhibition and senolytic therapy may delay the progression of kidney disease in NPHP type 7 and other forms of this disease. Nephronophthisis (NPHP), the leading genetic cause of end-stage renal failure in children and young adults, is a group of autosomal recessive diseases characterized by kidney-cyst degeneration and fibrosis for which no therapy is currently available. To date, mutations in >25 genes have been identified as causes of this disease that, in several cases, result in chronic DNA damage in kidney tubular cells. Among such mutations, those in the transcription factor–encoding GLIS2 cause NPHP type 7. Loss of function of mouse Glis2 causes senescence of kidney tubular cells. Senescent cells secrete proinflammatory molecules that induce progressive organ damage through several pathways, among which NF-κB signaling is prevalent. Herein, we show that the NF-κB signaling is active in Glis2 knockout kidney epithelial cells and that genetic inactivation of the toll-like receptor (TLR)/IL-1 receptor or pharmacologic elimination of senescent cells (senolytic therapy) reduces tubule damage, fibrosis, and apoptosis in the Glis2 mouse model of NPHP. Notably, in Glis2, Tlr2 double knockouts, senescence was also reduced and proliferation was increased, suggesting that loss of TLR2 activity improves the regenerative potential of tubular cells in Glis2 knockout kidneys. Our results further suggest that a combination of TLR/IL-1 receptor inhibition and senolytic therapy may delay the progression of kidney disease in NPHP type 7 and other forms of this disease. Nephronophthisis (NPHP), the most frequent monogenic cause of chronic renal failure during the first three decades of life, is a group of autosomal recessive diseases that are characterized by progressive cystic and fibrotic degeneration of the kidneys.1Wolf M.T. Nephronophthisis and related syndromes.Curr Opin Pediatr. 2015; 27: 201-211Crossref PubMed Scopus (83) Google Scholar From the genetic standpoint, NPHP is a heterogeneous disease, caused by mutations identified in >25 genes.2Luo F. Tao Y.H. Nephronophthisis: a review of genotype-phenotype correlation.Nephrology (Carlton). 2018; 23: 904-911Crossref PubMed Scopus (37) Google Scholar Despite the genetic heterogeneity of NPHP, almost all forms share two common progressive features: interstitial fibrosis and kidney atrophy. Chronic DNA damage in kidney tubular cells is common to at least six forms of NPHP, each caused by mutations in a distinct gene (ZNF423, CEP164, MRE11, NEK8, SDCCAG8, and CEP290).3Chaki M. Airik R. Ghosh A.K. Giles R.H. Chen R. Slaats G.G. et al.Exome capture reveals ZNF423 and CEP164 mutations, linking renal ciliopathies to DNA damage response signaling.Cell. 2012; 150: 533-548Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 4Choi H.J. Lin J.R. Vannier J.B. Slaats G.G. Kile A.C. Paulsen R.D. Manning D.K. Beier D.R. Giles R.H. Boulton S.J. Cimprich K.A. NEK8 links the ATR-regulated replication stress response and S phase CDK activity to renal ciliopathies.Mol Cell. 2013; 51: 423-439Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 5Airik R. Slaats G.G. Guo Z. Weiss A.C. Khan N. Ghosh A. Hurd T.W. Bekker-Jensen S. Schroder J.M. Elledge S.J. Andersen J.S. Kispert A. Castelli M. Boletta A. Giles R.H. Hildebrandt F. Renal-retinal ciliopathy gene Sdccag8 regulates DNA damage response signaling.J Am Soc Nephrol. 2014; 25: 2573-2583Crossref PubMed Scopus (51) Google Scholar, 6Slaats G.G. Saldivar J.C. Bacal J. Zeman M.K. Kile A.C. Hynes A.M. Srivastava S. Nazmutdinova J. den Ouden K. Zagers M.S. Foletto V. Verhaar M.C. Miles C. Sayer J.A. Cimprich K.A. Giles R.H. DNA replication stress underlies renal phenotypes in CEP290-associated Joubert syndrome.J Clin Invest. 2015; 125: 3657-3666Crossref PubMed Scopus (37) Google Scholar Loss of function of Glis2 causes DNA damage and activation of the replication stress response, resulting in extensive senescence of kidney epithelial cells in the mouse model of Nphp7.7Lu D. Rauhauser A. Li B. Ren C. McEnery K. Zhu J. Chaki M. Vadnagara K. Elhadi S. Jetten A.M. Igarashi P. Attanasio M. Loss of Glis2/NPHP7 causes kidney epithelial cell senescence and suppresses cyst growth in the Kif3a mouse model of cystic kidney disease.Kidney Int. 2016; 89: 1307-1323Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar This finding was of particular notice, providing a potential explanation for the link between DNA damage and progressive fibrosis in NPHP. Specifically, senescent cells are arrested in the cell cycle and secrete a wide spectrum of proinflammatory signaling molecules under the control of the proinflammatory and prosurvival pathway NF-κB,8Acosta J.C. Banito A. Wuestefeld T. Georgilis A. Janich P. Morton J.P. Athineos D. Kang T.W. Lasitschka F. Andrulis M. Pascual G. Morris K.J. Khan S. Jin H. Dharmalingam G. Snijders A.P. Carroll T. Capper D. Pritchard C. Inman G.J. Longerich T. Sansom O.J. Benitah S.A. Zender L. Gil J. A complex secretory program orchestrated by the inflammasome controls paracrine senescence.Nat Cell Biol. 2013; 15: 978-990Crossref PubMed Scopus (1161) Google Scholar, 9Tchkonia T. Zhu Y. van Deursen J. Campisi J. Kirkland J.L. Cellular senescence and the senescent secretory phenotype: therapeutic opportunities.J Clin Invest. 2013; 123: 966-972Crossref PubMed Scopus (1052) Google Scholar, 10Chien Y. Scuoppo C. Wang X. Fang X. Balgley B. Bolden J.E. Premsrirut P. Luo W. Chicas A. Lee C.S. Kogan S.C. Lowe S.W. Control of the senescence-associated secretory phenotype by NF-kappaB promotes senescence and enhances chemosensitivity.Genes Dev. 2011; 25: 2125-2136Crossref PubMed Scopus (593) Google Scholar causing what is known as the senescence-associated secretory phenotype (SASP). Notably, this activity can lead to the induction of senescence in nonsenescent bystander cells, resulting in a loop that amplifies inflammation and organ damage.11Campisi J. Cellular senescence: putting the paradoxes in perspective.Curr Opin Genet Dev. 2011; 21: 107-112Crossref PubMed Scopus (270) Google Scholar,12Herranz N. Gil J. Mechanisms and functions of cellular senescence.J Clin Invest. 2018; 128: 1238-1246Crossref PubMed Scopus (464) Google Scholar The inflammatory cytokine IL-1α is of particular importance in mediating this phenomenon. It signals through its cognate receptor IL-1 receptor (IL-1R),13Orjalo A.V. Bhaumik D. Gengler B.K. Scott G.K. Campisi J. Cell surface-bound IL-1alpha is an upstream regulator of the senescence-associated IL-6/IL-8 cytokine network.Proc Natl Acad Sci U S A. 2009; 106: 17031-17036Crossref PubMed Scopus (418) Google Scholar which is a member of the toll-like receptor (TLR)/IL-1R family. Like other members of this receptor family, such as TLR2 and TLR4, which are ubiquitously expressed, including in kidney epithelial cells, on stimulation IL-1R activates the NF-κB pathway via common cellular adapter proteins, among which is myeloid differentiation primary response 88 (Myd88).14Kawai T. Akira S. Signaling to NF-kappaB by Toll-like receptors.Trends Mol Med. 2007; 13: 460-469Abstract Full Text Full Text PDF PubMed Scopus (1638) Google Scholar,15Ve T. Gay N.J. Mansell A. Kobe B. Kellie S. Adaptors in toll-like receptor signaling and their potential as therapeutic targets.Curr Drug Targets. 2012; 13: 1360-1374Crossref PubMed Scopus (69) Google Scholar Multiple components of the SASP interact with receptors of the innate immune system, including IL-1R (IL-1α/IL-1β), TLR4, and TLR2 (high-mobility group protein 1; other alarmins). Among the consequences of NF-κB activation is the transcription of the TLR2 gene itself, resulting in amplification of the NF-κB signaling.16Johnson C.M. Tapping R.I. Microbial products stimulate human Toll-like receptor 2 expression through histone modification surrounding a proximal NF-kappaB-binding site.J Biol Chem. 2007; 282: 31197-31205Crossref PubMed Scopus (39) Google Scholar,17Coppe J.P. Desprez P.Y. Krtolica A. Campisi J. The senescence-associated secretory phenotype: the dark side of tumor suppression.Annu Rev Pathol. 2010; 5: 99-118Crossref PubMed Scopus (2638) Google Scholar Given that DNA damage is known to activate NF-κB,18Janssens S. Tschopp J. Signals from within: the DNA-damage-induced NF-kappaB response.Cell Death Differ. 2006; 13: 773-784Crossref PubMed Scopus (254) Google Scholar,19McCool K.W. Miyamoto S. DNA damage-dependent NF-kappaB activation: NEMO turns nuclear signaling inside out.Immunol Rev. 2012; 246: 311-326Crossref PubMed Scopus (176) Google Scholar we hypothesized that unprogrammed nuclear events triggered by loss-of-function mutations in Glis2 might be the primary causes of NF-κB activation in kidney epithelial cells and that such activation might be amplified by subsequent increases in signaling via IL-1R, TLR2, and TLR4. We examined the contribution of TLRs/IL-1R signaling to kidney damage, inflammation, and senescence in Glis2 knockout mice. Two mouse strains were generated: a double-knockout strain lacking both Glis2 and Tlr2 in all tissues (Glis2−/−;Tlr2−/−) and a strain in which a kidney-specific promoter (Ksp)20Shao X. Somlo S. Igarashi P. Epithelial-specific Cre/lox recombination in the developing kidney and genitourinary tract.J Am Soc Nephrol. 2002; 13: 1837-1846Crossref PubMed Scopus (235) Google Scholar is used to inactivate Myd88 in tubular cells of Glis2-null mice (Glis2−/−;KspCreMyd88f/f). Both constitutive inactivation of Tlr2 and tubular cell–specific conditional deletion of Myd88 were associated with reduced kidney damage, fibrosis, DNA damage, and apoptosis in Glis2 knockout mice. Moreover, in the double-knockout mice, tubular cell senescence was decreased and proliferation was increased. Pharmacologic elimination of senescent cells in Glis2 knockout mice resulted in a similar outcome in terms of kidney damage, fibrosis, and apoptosis, but cell senescence and proliferation were not affected. The immortalized kidney epithelial cell lines that were stably transduced with inducible Glis2-targeting shRNA or the shRNA targeting green fluorescent protein (control) are described elsewhere.7Lu D. Rauhauser A. Li B. Ren C. McEnery K. Zhu J. Chaki M. Vadnagara K. Elhadi S. Jetten A.M. Igarashi P. Attanasio M. Loss of Glis2/NPHP7 causes kidney epithelial cell senescence and suppresses cyst growth in the Kif3a mouse model of cystic kidney disease.Kidney Int. 2016; 89: 1307-1323Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar The efficiency of the Glis2 knockdown was confirmed by quantitative real-time PCR (>80%). RNA was extracted from the induced Glis2-targeting shRNA line and shRNA targeting green fluorescent protein using Trizol (Sigma, St. Louis, MO), incubated with DNAse for 1 hour, and used to probe the Illumina Mouse WG-6_V2_0_R0_11278593_A beadchip (Illumina, San Diego, CA), according to the manufacturer's instructions. Ingenuity software version 8.6 (Qiagen Bioinformatics, Hilden, Germany) was used for gene ontology analysis. Raw data are retrievable on the Gene Expression Omnibus website (https://www.ncbi.nlm.nih.gov/geo; accession number GSE134338). All mice were on a C57/B6 background. The Glis2−/− mice were previously described.21Kim Y.S. Kang H.S. Herbert R. Beak J.Y. Collins J.B. Grissom S.F. Jetten A.M. Kruppel-like zinc finger protein Glis2 is essential for the maintenance of normal renal functions.Mol Cell Biol. 2008; 28: 2358-2367Crossref PubMed Scopus (52) Google Scholar The KspCre mice20Shao X. Somlo S. Igarashi P. Epithelial-specific Cre/lox recombination in the developing kidney and genitourinary tract.J Am Soc Nephrol. 2002; 13: 1837-1846Crossref PubMed Scopus (235) Google Scholar were obtained from the O'Brien Kidney Center at the UT Southwestern Medical Center (Dallas, TX). Tlr2 knockout mice and Myd88fl/fl conditional mice were purchased from the Jax Laboratories (Bar Harbor, ME; stock numbers 4650 and 8888, respectively). All procedures were approved by the Institutional Animal Care and Use Committee of the University of Iowa (Iowa City, IA). The NF-κB pathway sampler kit (9936) was obtained from Cell Signaling (Danvers, MA), and antibodies were obtained from the following sources: anti-CD68 (MCA1957T) from Bio-Rad (Hercules, CA), anti-TLR2 (T2.5) from Invivogen (San Diego, CA), anti–α-smooth muscle actin (A5228) from Sigma, anti–cleaved caspase-3 (AB3623MI) from Thermo Fisher Scientific (Waltham, MA), and anti-fibroblast specific protein 1 (FSP1; ab27957) and anti–Ki-67 (ab15580) from Abcam (Cambridge, MA). Kidney sections were stained with periodic acid–Schiff by the pathology core at UT Southwestern Medical Center or at the University of Iowa; and for each sample, seven consecutive images were taken using a Zeiss (Oberkochen, Germany) Axioplan 2 deconvolution microscope. Tubule injury scores were assigned to periodic acid–Schiff–stained kidney sections (4 μm thick) by an experienced kidney pathologist (P.R.) who was blinded to the identity of the samples. Published criteria were adopted, as described, for each parameter.22Hur E. Garip A. Camyar A. Ilgun S. Ozisik M. Tuna S. Olukman M. Narli Ozdemir Z. Yildirim Sozmen E. Sen S. Akcicek F. Duman S. The effects of vitamin d on gentamicin-induced acute kidney injury in experimental rat model.Int J Endocrinol. 2013; 2013: 313528Crossref PubMed Scopus (41) Google Scholar Indicators of tubule injury/degeneration were vacuolization, intraluminal casts, and acellular/atrophic tubules. Scoring was as follows: 0 indicates none detected; 1, 1% to 10% of tubules affected; 2, 11% to 25% of tubules affected; 3, 26% to 50% of tubules affected; and 4, >50% of tubules affected. Tubulointerstitial inflammation was defined by the presence of lymphocytes in perivascular and interstitial cortical areas. Scoring was as follows: 0 indicates no significant inflammation; 1, foci in 1% to 10% of perivascular areas; 2, 11% to 25% of the cortex affected; 3, 26% to 50% of the cortex affected; and 4, >50% of the cortex affected. Serum was obtained as the supernatant from blood samples centrifuged at 1500 × g for 10 minutes at room temperature. The serum urea concentration was determined using the Urea Assay Kit from Abcam (ab83362). Ten paraffin-embedded kidney sections (10 μm thick) per mouse were hydrolyzed in 150 mL 6 mol/L HCl, incubated for 20 hours at 95°C, cooled, and centrifuged for 10 minutes at 16,000 × g. Concentrations of collagen and total protein in the supernatant were determined using collagen and protein assay kits from QuickZyme Biosciences (Leiden, the Netherlands). Kidneys were collected after mice were perfused with phosphate-buffered saline containing 4% paraformaldehyde, then fixed in 4% paraformaldehyde at 4°C overnight, left in 18% sucrose solution in phosphate-buffered saline at 4°C overnight, and embedded in OCT compound. Frozen tissue sections (10 μm thick) were air dried for 30 minutes at room temperature and sequentially incubated in 0.1% Triton X-100 solution in phosphate-buffered saline (20 minutes), 0.1% NaBH4 solution (30 minutes), and blocking solution (1 hour) at room temperature. Tissues were incubated with primary antibody diluted in blocking solution overnight at 4°C and then with fluorescently labeled secondary antibody for 1 hour at room temperature. Samples were mounted using ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific, Waltham, MA). Images were acquired using either a Zeiss Axioplan 2 deconvolution microscope or a Zeiss LSM 710 confocal microscope with constant acquisition parameters. Kidneys were collected and prepared as described above for immunofluorescence staining. Frozen sections (8 μm thick) were air dried for 20 minutes and then incubated at 37°C for 12 to 16 hours in fresh senescence-associated β galactosidase (SA-β-Gal) staining solution, which contains 1 mg/mL X-Gal (stock solution in dimethylformamide; Teknova, Hollister, CA), 40 mmol/L citric acid/sodium phosphate (pH 6.0), 5 mmol/L potassium ferrocyanide, 5 mmol/L potassium ferricyanide, 150 mmol/L NaCl, and 2 mmol/L MgCl2. Sections were counterstained with eosin. Images of Western blot analyses, kidney sections stained using a protocol for immunofluorescence, SA-β-Gal, or Masson's trichrome were subjected to quantitative digital analysis. For each cortical region, 10 consecutive ×200 optical fields per section were selected for measurement. Signal was quantified by digital image analysis using ImageJ software version 1.52c (NIH, Bethesda, MD; http://imagej.nih.gov/ij). Total RNA was extracted using the Qiagen RNeasy Mini kit. RNA was reverse transcribed using the iScript cDNA Synthesis Kit (Bio-Rad). Real-time PCR was performed using the CFX Connect Real-Time PCR Detection System (Bio-Rad) and iTaq Universal SYBR Green Supermix (Bio-Rad). All real-time PCR experiments were performed in triplicate. PCR primers were obtained from Integrated DNA Technology (Coralville, IA). Their sequences are the following: β-actin, 5′-GTACCACCATGTACCCAGGC-3′ (forward) and 5′-AACGCAGCTCAGTAACAGTC-3′ (reverse); IL-1α, 5′-CGAAGACTACAGTTCTGCCATT-3′ (forward) and 5′-GACGTTTCAGAGGTTCTCAGAG-3′ (reverse); IL-1β, 5′-GCAACTGTTCCTGAACTCAACT-3′ (forward) and 5′-ATCTTTTGGGGTCCGTCAACT-3′ (reverse); and tumor necrosis factor-1α, 5′-GCCTCTTCTCATTCCTGCTTG-3′ (forward) and 5′-CTGATGAGAGGGAGGCCATT-3′ (reverse). All data are presented as means ± SD. Statistical significance (P ≤ 0.05 was considered significant) was calculated using the two-tailed t-test or analysis of variance and Tukey's multiple comparisons test for more than two groups, using the SPSS17.0 software (IBM, Armonk, NY) or GraphPad Prism 7 (GraphPad Software, San Diego, CA). To gain insights into the molecular consequences of Glis2 loss in kidney epithelial cells, microarray-based differential expression analysis was performed, comparing immortalized mouse tubular cells stably expressing a Glis2-targeting shRNA.23Li B. Rauhauser A.A. Dai J. Sakthivel R. Igarashi P. Jetten A.M. Attanasio M. Increased hedgehog signaling in postnatal kidney results in aberrant activation of nephron developmental programs.Hum Mol Genet. 2011; 20: 4155-4166Crossref PubMed Scopus (31) Google Scholar Counterpart cells stably expressing a shRNA targeting green fluorescent protein were used as a control. Many genes encoding components of the NF-κB pathway, including Tlr2, were up-regulated in the Glis2-silenced but not control cells, suggesting that NF-κB signaling is active in the absence of Glis2 (Figure 1A). Western blot analyses of cell lysates revealed that phosphorylation of serine 563 of the NF-κB subunit p65, as well as of serine 32 of IκBα, was higher in the Glis2-silenced versus control cells, confirming that the NF-κB pathway was active (Figure 1, B and C). Supplementation of the culture medium with the TLR2 ligand lipopolysaccharide neither induced significant p65 phosphorylation in control cells nor further increased p65 phosphorylation in Glis2 knockdown cells. However, addition of a TLR2-blocking antibody (T2.5) resulted in a clear reduction of p65 phosphorylation in Glis2 knockdown cells (Figure 1D). These findings indicate that Tlr2 contributes to NF-κB activation in kidney tubular cells deficient for Glis2. To assess the physiological relevance of these results, the consequences of loss of Glis2 were tested in vivo, comparing Tlr2 activation in the kidney epithelial cells of the Glis2 knockout mouse with that in wild-type control animals. Examination of tubules within kidney sections by immunofluorescence confocal microscopy after staining with an antibody against Tlr2 revealed that, although in control samples the signal was localized at cell-cell junctions as expected, in the tubules of Glis2 knockout mice it was present in puncta throughout the cytoplasm (Figure 1, E and F). This staining pattern is consistent with ligand-bound Tlr2 being activated and endocytosed in the Glis2 knockout tubular cells in vivo.24Brandt K.J. Fickentscher C. Kruithof E.K. de Moerloose P. TLR2 ligands induce NF-kappaB activation from endosomal compartments of human monocytes.PLoS One. 2013; 8: e80743Crossref PubMed Scopus (50) Google Scholar Collectively, these results indicate that the NF-κB signaling pathway is activated in Glis2 knockout kidney epithelial cells and that such activation is reinforced by signaling downstream of TLR2. On the basis of the results shown in Figure 1, we hypothesized that inactivation of signaling downstream of Tlr2 might ameliorate the kidney phenotype of the Glis2 knockout mice. To test this hypothesis, Glis2−/−;Tlr2−/− double-knockout mice were generated and the resulting phenotype was compared with that of the Glis2 knockout mice. This analysis was performed when the mice were 3 months of age, by which time point kidney damage and fibrosis have clearly manifested.21Kim Y.S. Kang H.S. Herbert R. Beak J.Y. Collins J.B. Grissom S.F. Jetten A.M. Kruppel-like zinc finger protein Glis2 is essential for the maintenance of normal renal functions.Mol Cell Biol. 2008; 28: 2358-2367Crossref PubMed Scopus (52) Google Scholar,25Attanasio M. Uhlenhaut N.H. Sousa V.H. O'Toole J.F. Otto E. Anlag K. Klugmann C. Treier A.C. Helou J. Sayer J.A. Seelow D. Nurnberg G. Becker C. Chudley A.E. Nurnberg P. Hildebrandt F. Treier M. Loss of GLIS2 causes nephronophthisis in humans and mice by increased apoptosis and fibrosis.Nat Genet. 2007; 39: 1018-1024Crossref PubMed Scopus (180) Google Scholar Analysis of bright-field microscopy images of kidney sections stained with periodic acid–Schiff (Figure 2A) revealed that tubule morphology (Figure 2B) and cystic dilation (Figure 2C) were less severe in the double-knockout versus control mice. Renal function, assessed on the basis of serum urea concentrations, was also better in the double-knockout mice (Figure 2D). Examination of kidney sections by Masson's trichrome protocol (Figure 2E) showed that deletion of Tlr2 in mice deficient for Glis2 also protected against fibrosis (Figure 2, F and G), and digital quantification of immunofluorescence confocal microscopy images revealed that it prevented interstitial accumulation of fibroblasts (FSP-1–positive cells) and macrophages (CD68-positive cells) (Figure 2, H–K). The expression of IL-1α and tumor necrosis factor, but not IL-1β (Figure 2, L–N), was also lower in the double-knockout versus Glis2 knockout mice, indicating that a block of signaling downstream of Tlr2 prevents tubule damage, inflammation, and fibrosis that characterizes kidneys of the Glis2 knockout mice. Loss of Glis2 in kidney tubule cells causes activation of the replication stress response and extensive cell senescence.7Lu D. Rauhauser A. Li B. Ren C. McEnery K. Zhu J. Chaki M. Vadnagara K. Elhadi S. Jetten A.M. Igarashi P. Attanasio M. Loss of Glis2/NPHP7 causes kidney epithelial cell senescence and suppresses cyst growth in the Kif3a mouse model of cystic kidney disease.Kidney Int. 2016; 89: 1307-1323Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar In senescent cells, activation of the NF-κB pathway induces secretion of a wide array of proinflammatory molecules, including IL-1α, which signals through IL-1R on the recipient cells to promote senescence in the neighboring cells.8Acosta J.C. Banito A. Wuestefeld T. Georgilis A. Janich P. Morton J.P. Athineos D. Kang T.W. Lasitschka F. Andrulis M. Pascual G. Morris K.J. Khan S. Jin H. Dharmalingam G. Snijders A.P. Carroll T. Capper D. Pritchard C. Inman G.J. Longerich T. Sansom O.J. Benitah S.A. Zender L. Gil J. A complex secretory program orchestrated by the inflammasome controls paracrine senescence.Nat Cell Biol. 2013; 15: 978-990Crossref PubMed Scopus (1161) Google Scholar, 9Tchkonia T. Zhu Y. van Deursen J. Campisi J. Kirkland J.L. Cellular senescence and the senescent secretory phenotype: therapeutic opportunities.J Clin Invest. 2013; 123: 966-972Crossref PubMed Scopus (1052) Google Scholar, 10Chien Y. Scuoppo C. Wang X. Fang X. Balgley B. Bolden J.E. Premsrirut P. Luo W. Chicas A. Lee C.S. Kogan S.C. Lowe S.W. Control of the senescence-associated secretory phenotype by NF-kappaB promotes senescence and enhances chemosensitivity.Genes Dev. 2011; 25: 2125-2136Crossref PubMed Scopus (593) Google Scholar Because the same signaling mechanisms result in activation of NF-κB downstream of both TLR2 and IL-1R, and these are further shared by TLR4, we hypothesized that the TLR/IL-1R signaling axis is responsible for a spread of the senescent phenotype in our mouse model of NPHP type 7. Our comparison of kidneys of the double-knockout versus Glis2 knockout mice showed that the activity of SA-β-Gal in tubules was lower in the double-knockout versus Glis2 knockout kidneys (Figure 3, A and B ). Consistent with this finding, the ratio of cells positive for the DNA damage marker γH2AX was lower, and that of the cells, tubular and interstitial, positive for the proliferation marker Ki-67 was higher in the double-knockout versus Glis2 knockout mice (Figure 3, C–F). The number of apoptotic cells, a feature of the Glis2 knockout kidney phenotype,25Attanasio M. Uhlenhaut N.H. Sousa V.H. O'Toole J.F. Otto E. Anlag K. Klugmann C. Treier A.C. Helou J. Sayer J.A. Seelow D. Nurnberg G. Becker C. Chudley A.E. Nurnberg P. Hildebrandt F. Treier M. Loss of GLIS2 causes nephronophthisis in humans and mice by increased apoptosis and fibrosis.Nat Genet. 2007; 39: 1018-1024Crossref PubMed Scopus (180) Google Scholar was also lower in the double-knockout kidneys (Figure 3, G and H). Tlr2, Tlr4, and IL-1R all signal through common downstream molecules, including Myd88. Tubule-specific deletion of Myd88 was sufficient to improve kidney damage, fibrosis, and tubular cell senescence in a mouse model of acute kidney injury.26Jin H. Zhang Y. Ding Q. Wang S.S. Rastogi P. Dai D.F. Lu D. Purvis M. Cao C. Wang A. Liu D. Ren C. Elhadi S. Hu M.C. Chai Y. Zepeda-Orozco D. Campisi J. Attanasio M. Epithelial innate immunity mediates tubular cell senescence after kidney injury.JCI Insight. 2019; 4 (e125490)Crossref Scopus (57) Google Scholar To determine whether the inactivation of Myd88 in epithelial cells would ameliorate the kidney phenotype of the Glis2 knockout, Glis2 knockout mice were generated in which both copies of the conditional allele Myd88 were knocked out in kidney tubular cells. The kidney-specific (KspCre) transgenic strain, in which Cre expression is driven by the promoter of the tubular cell–specific gene cadherin 16, was used.20Shao X. Somlo S. Igarashi P. Epithelial-specific Cre/lox recombination in the developing kidney and genitourinary tract.J Am Soc Nephrol. 2002; 13: 1837-1846Crossref PubMed Scopus (235) Google Scholar As in the case of our Glis2, Tlr2 double-knockout mice, the kidneys of Glis2−/−;KspCreMyd88f/f mice had fewer signs of tubule damage: in images of sections of Glis2−/−;KspCreMyd88f/f kidneys stained with periodic acid–Schiff, fewer tubules were damaged, kidney atrophy was less pronounced, and the number of glomeruli with a dilated Bowman capsule was smaller (Figure 4, A–C). Serum concentrations of urea were also lower (Figure 4D). Concordant with this finding, collagen infiltration, as assessed by digital analysis of bright-field microscopy images of Masson's trichrome–stained kidneys, as well as the ratio of collagen/total protein were lower in Glis2−/−;KspCreMyd88f/f than Glis2 single-knockout mice (Figure 4, E–G). However, the number of interstitial fibroblasts and macrophages (Figure 4, H–K), as well as the levels of the inflammatory cytokines IL-1α, IL-1β, and tumor necrosis factor-α (Figure 4, L–N), did not differ, suggesting that epithelium-specific deletion of Myd88 is only partially protective against the Glis2 knockout kidney phenotype. To determine whether inactivation of Myd88 in tubular cells affects their senescence, the activity of SA-β-Gal was estimated and immunofluorescence microscopy was performed on kidney sections of Glis2−/−;KspCreMyd88f/f and Glis2 knockout mice using antibodies against Ki-67, a marker of actively cycling cells. Unlike the kidneys of the Glis2, Tlr2 double-knockout mice, those of the Glis2−/−;KspCreMyd88f/f mice did not differ from those from Glis2 knockout kidneys in terms of SA-β-Gal activity, although the number of proliferating cells was slightly higher in the former (Figure 5, A and B ). However, DNA damage (Figure 5, C and D) was lower in Glis2−/−;KspCreMyd88f/f versus Glis2 knockout kidneys. The number of proliferating cells (Figure 5, E and F) was slightly higher in the former, and the number of apoptotic cells (Figure 5, G and H) was s
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