BCL‐XL exerts a protective role against anemia caused by radiation‐induced kidney damage
2020; Springer Nature; Volume: 39; Issue: 24 Linguagem: Inglês
10.15252/embj.2020105561
ISSN1460-2075
AutoresKerstin Brinkmann, Paul Waring, Stefan Glaser, Verena C. Wimmer, Denny L. Cottle, Ming Shen Tham, Duong Nhu, Lachlan Whitehead, Alex R. D. Delbridge, Guillaume Lessène, Ian Smyth, Marco J. Herold, Gemma L. Kelly, Stephanie Grabow, Andreas Strasser,
Tópico(s)Cell death mechanisms and regulation
ResumoArticle25 November 2020free access Source DataTransparent process BCL-XL exerts a protective role against anemia caused by radiation-induced kidney damage Kerstin Brinkmann Corresponding Author [email protected] orcid.org/0000-0002-9411-6674 The Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic., Australia Department of Medical Biology, University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Paul Waring Department of Surgery, University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Stefan P Glaser The Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic., Australia Department of Medical Biology, University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Verena Wimmer The Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic., Australia Department of Medical Biology, University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Denny L Cottle Department of Anatomy and Developmental Biology, Development and Stem Cell Program Monash Biomedicine Discovery Institute (BDI), Monash University, Melbourne, Vic., Australia Search for more papers by this author Ming Shen Tham Department of Anatomy and Developmental Biology, Development and Stem Cell Program Monash Biomedicine Discovery Institute (BDI), Monash University, Melbourne, Vic., Australia Search for more papers by this author Duong Nhu The Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic., Australia Department of Medical Biology, University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Lachlan Whitehead The Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic., Australia Department of Medical Biology, University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Alex RD Delbridge The Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic., Australia Department of Medical Biology, University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Guillaume Lessene orcid.org/0000-0002-1193-8147 The Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic., Australia Department of Medical Biology, University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Ian M Smyth orcid.org/0000-0002-1727-7829 Department of Anatomy and Developmental Biology, Development and Stem Cell Program Monash Biomedicine Discovery Institute (BDI), Monash University, Melbourne, Vic., Australia Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Vic., Australia Search for more papers by this author Marco J Herold orcid.org/0000-0001-7539-7581 The Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic., Australia Department of Medical Biology, University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Gemma L Kelly The Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic., Australia Department of Medical Biology, University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Stephanie Grabow The Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic., Australia Department of Medical Biology, University of Melbourne, Melbourne, Vic., Australia These authors are contributed equally to this worked as senior authors Search for more papers by this author Andreas Strasser Corresponding Author [email protected] orcid.org/0000-0002-5020-4891 The Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic., Australia Department of Medical Biology, University of Melbourne, Melbourne, Vic., Australia These authors are contributed equally to this worked as senior authors Search for more papers by this author Kerstin Brinkmann Corresponding Author [email protected] orcid.org/0000-0002-9411-6674 The Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic., Australia Department of Medical Biology, University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Paul Waring Department of Surgery, University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Stefan P Glaser The Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic., Australia Department of Medical Biology, University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Verena Wimmer The Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic., Australia Department of Medical Biology, University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Denny L Cottle Department of Anatomy and Developmental Biology, Development and Stem Cell Program Monash Biomedicine Discovery Institute (BDI), Monash University, Melbourne, Vic., Australia Search for more papers by this author Ming Shen Tham Department of Anatomy and Developmental Biology, Development and Stem Cell Program Monash Biomedicine Discovery Institute (BDI), Monash University, Melbourne, Vic., Australia Search for more papers by this author Duong Nhu The Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic., Australia Department of Medical Biology, University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Lachlan Whitehead The Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic., Australia Department of Medical Biology, University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Alex RD Delbridge The Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic., Australia Department of Medical Biology, University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Guillaume Lessene orcid.org/0000-0002-1193-8147 The Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic., Australia Department of Medical Biology, University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Ian M Smyth orcid.org/0000-0002-1727-7829 Department of Anatomy and Developmental Biology, Development and Stem Cell Program Monash Biomedicine Discovery Institute (BDI), Monash University, Melbourne, Vic., Australia Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Vic., Australia Search for more papers by this author Marco J Herold orcid.org/0000-0001-7539-7581 The Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic., Australia Department of Medical Biology, University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Gemma L Kelly The Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic., Australia Department of Medical Biology, University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Stephanie Grabow The Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic., Australia Department of Medical Biology, University of Melbourne, Melbourne, Vic., Australia These authors are contributed equally to this worked as senior authors Search for more papers by this author Andreas Strasser Corresponding Author [email protected] orcid.org/0000-0002-5020-4891 The Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic., Australia Department of Medical Biology, University of Melbourne, Melbourne, Vic., Australia These authors are contributed equally to this worked as senior authors Search for more papers by this author Author Information Kerstin Brinkmann *,1,2, Paul Waring3, Stefan P Glaser1,2,†, Verena Wimmer1,2, Denny L Cottle4, Ming Shen Tham4, Duong Nhu1,2, Lachlan Whitehead1,2, Alex RD Delbridge1,2,†, Guillaume Lessene1,2, Ian M Smyth4,5, Marco J Herold1,2, Gemma L Kelly1,2, Stephanie Grabow1,2,† and Andreas Strasser *,1,2 1The Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic., Australia 2Department of Medical Biology, University of Melbourne, Melbourne, Vic., Australia 3Department of Surgery, University of Melbourne, Melbourne, Vic., Australia 4Department of Anatomy and Developmental Biology, Development and Stem Cell Program Monash Biomedicine Discovery Institute (BDI), Monash University, Melbourne, Vic., Australia 5Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Vic., Australia †Present address: Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria †Present address: Putnam Associates, Boston, MA, USA †Present address: Blueprint Medicines, Cambridge, MA, USA *Corresponding author. Tel: +61 3 9345 2555; E-mail: [email protected] *Corresponding author. Tel: +61 3 9345 2555; E-mail: [email protected] EMBO J (2020)39:e105561https://doi.org/10.15252/embj.2020105561 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Studies of gene-targeted mice identified the roles of the different pro-survival BCL-2 proteins during embryogenesis. However, little is known about the role(s) of these proteins in adults in response to cytotoxic stresses, such as treatment with anti-cancer agents. We investigated the role of BCL-XL in adult mice using a strategy where prior bone marrow transplantation allowed for loss of BCL-XL exclusively in non-hematopoietic tissues to prevent anemia caused by BCL-XL deficiency in erythroid cells. Unexpectedly, the combination of total body γ-irradiation (TBI) and genetic loss of Bcl-x caused secondary anemia resulting from chronic renal failure due to apoptosis of renal tubular epithelium with secondary obstructive nephropathy. These findings identify a critical protective role of BCL-XL in the adult kidney and inform on the use of BCL-XL inhibitors in combination with DNA damage-inducing drugs for cancer therapy. Encouragingly, the combination of DNA damage-inducing anti-cancer therapy plus a BCL-XL inhibitor could be tolerated in mice, at least when applied sequentially. SYNOPSIS The pro-survival BCL-2 family member BCL-XL is critical for the survival of renal proximal tubular epithelial cells during radiation therapy in cancer patients. Genetic loss of BCL-XL, but not its pharmacological inhibition, causes fatal kidney damage and secondary anemia in adult mice after total body irradiation. Inducible loss of BCL-XL in all cells in adult mice causes primary anemia due to apoptosis of erythroid and megakaryocytic cell populations. γ-radiation and BCL-XL deficiency in all non-hematopoietic cells cause secondary anemia resulting from kidney damage. γ-radiation or DNA damage-inducing drugs in combination with specific BCL-XL inhibitor A1331852 is tolerated in mice. Introduction Evasion of apoptotic cell death is a hallmark of cancer (Hanahan & Weinberg, 2000, 2011), and direct activation of the cell death machinery by BH3-mimetic drugs represents an attractive therapeutic strategy (Merino et al, 2018). Apoptosis is controlled by pro- and anti-apoptotic members of the BCL-2 protein family (Tait & Green, 2010), with the transcriptional or post-transcriptional activation of the pro-apoptotic BH3-only members of the BCL-2 family (e.g., BIM, PUMA) being its initiator. The BH3-only proteins bind with high affinity to the anti-apoptotic BCL-2 family members (e.g., BCL-2, BCL-XL), thereby unleashing the pro-apoptotic effector proteins BAX and BAK to cause mitochondrial outer membrane permeabilization (MOMP). MOMP causes the release of activators of the caspase cascade that causes cell demolition (Riedl & Salvesen, 2007; Tait & Green, 2010). The extent of MOMP and apoptosis is considered key determinants of therapeutic success of anti-cancer therapeutics (Del Gaizo Moore & Letai, 2013). BH3-only proteins, particularly PUMA (Villunger et al, 2003) and BIM (Bouillet et al, 1999a), are critical for the killing of malignant (and non-transformed) cells by diverse anti-cancer agents. As a consequence of genetic or epigenetic changes, many cancers show a reduction in pro-apoptotic BH3-only proteins or overexpression of pro-survival BCL-2 proteins (Uhlen et al, 2005; Beroukhim et al, 2010; Uhlen et al, 2017). Certain cancer cells can be killed by the loss or inhibition of a single pro-survival BCL-2 family member (Glaser et al, 2012; Kelly et al, 2014), whereas others require loss/inhibition of two or more of these proteins (Campbell & Tait, 2018). Several small-molecule inhibitors of pro-survival BCL-2 proteins (BH3-mimetic drugs) have been developed (Adams & Cory, 2018). Even though BH3-mimetics can potently kill diverse cancer cells in vitro and in vivo, the safe clinical use of some of these drugs remains challenging due to the obligate roles of pro-survival BCL-2 proteins for the survival of normal cells in healthy tissues (Adams & Cory, 2018). Gene deletion studies in mice have helped identify the critical roles of the different pro-survival BCL-2 family members. Loss of A1/BFL-1 causes minor reductions in certain hematopoietic cell subsets (Schenk et al, 2017; Tuzlak et al, 2017), and loss of BCL-W causes male sterility (Print et al, 1998). BCL-2-deficient mice die soon after weaning due to polycystic kidney disease and also present with reductions in mature B and T lymphocytes and melanocytes (causing premature graying) (Veis et al, 1993). These abnormalities could be prevented by concomitant loss of pro-apoptotic BIM (Bouillet et al, 2001). Constitutive loss of MCL-1 causes embryonic lethality prior to implantation (E3.5) (Rinkenberger et al, 2000), and tissue-restricted deletion revealed that many cell types, including cardiomyocytes (Thomas et al, 2013; Wang et al, 2013), neurons (Arbour et al, 2008), and several hematopoietic cell subsets (Opferman et al, 2005) require MCL-1 for survival. Loss of Bcl-x causes embryonic lethality at ~ E13.5 with aberrant death of neurons and immature hematopoietic cells (Motoyama et al, 1995). Conditional deletion of Bcl-x only in erythroid cells causes fatal anemia (Wagner et al, 2000), and loss of just one allele of Bcl-x impairs male fertility (Rucker et al, 2000) and reduces platelet numbers (Mason et al, 2007). BH3-mimetic drugs that selectively target BCL-XL (A1331852) (Lessene et al, 2013) or BCL-2 (ABT-199/venetoclax) (Souers et al, 2013) or compounds that inhibit BCL-XL, BCL-2, and BCL-W (ABT-737, ABT-263) (Oltersdorf et al, 2005; Tse et al, 2008) have been developed. Even though inhibitors of BCL-XL can efficiently kill diverse cancer cells, by themselves or in combination with additional anti-cancer agents (Oltersdorf et al, 2005; Cragg et al, 2007; Cragg et al, 2008; Tse et al, 2008), these compounds are progressing slowly in the clinic. This is in part due to the fact that the essential roles of BCL-XL in the adult (beyond its role in hematopoietic cells) are not clearly understood. Here we investigated the impact of inducible loss of BCL-XL in adult mice. This revealed that BCL-XL loss is tolerated throughout the body as long as the hematopoietic cells remain BCL-XL-sufficient. However, mice that had been γ-irradiated (for bone marrow transplantation) and then subjected to loss of BCL-XL developed secondary anemia due to chronic renal failure with secondary obstructive nephropathy characterized by renal tubular epithelial cell apoptosis. This morbidity could be markedly delayed, although not abrogated, by the concomitant loss of pro-apoptotic PUMA or BIM. These findings predict potential toxicities and their underlying mechanisms resulting from combinations of BCL-XL inhibitors with DNA damage-inducing anti-cancer therapeutics. Results Impact of induced deletion of BCL-XL in adult mice Bcl-x−/− embryos die ~ E13.5 due to loss of neuronal and erythroid cells (Motoyama et al, 1995). Mice with hypomorphic mutations in the Bcl-x gene are viable but develop severe thrombocytopenia and anemia (Mason et al, 2007). We wanted to explore the consequences of inducible loss of BCL-XL in adult mice. For this, we crossed Bcl-xfl/fl mice with RosaCreERT2 mice that express a CreERT2 fusion protein in all tissues. CreERT2 is normally kept inactive in the cytoplasm by binding to HSP90 but can be activated by tamoxifen (Vooijs et al, 2001). Even though BCL-XL is essential for neuronal cell survival (Motoyama et al, 1995), we did not expect any neuronal abnormalities upon tamoxifen treatment in Bcl-xfl/fl;RosaCreERT2+/Ki mice since tamoxifen administered by oral gavage did not efficiently drive CreERT2-mediated deletion of the Bcl-x-floxed genes in the brain, as shown by Southern blotting (Appendix Fig S1A). Almost complete deletion of the Bcl-x gene was achieved in most other tissues, including the spleen, kidney, liver, pancreas, intestine, and lung, whereas deletion in the testis was ~ 70%. As reported (Motoyama et al, 1995; Wagner et al, 2000), we found that induced loss of BCL-XL in adult mice caused fatal thrombocytopenia and anemia with a median survival of ~ 25 days (Fig 1A). Accordingly, the BCL-XL-deleted mice presented with significant decreases in platelets, red blood cells (RBCs), hemoglobin content (HGB), and hematocrit (HCT) (Fig 1C–F), accompanied by massively enlarged spleens due to compensatory erythropoiesis in this tissue (Fig 1G–I). Figure 1. The inducible deletion of BCL-XL causes severe anemia even in chimeric mice that are BCL-XL sufficient in all hematopoietic cell populations A. Bcl-xfl/fl;RosaCreERT2+/Ki (n = 12) or, as controls, Bcl-xfl/fl (n = 6), and RosaCreERT2+/Ki (n = 16) mice (age 9–12 weeks, males and females) were treated with tamoxifen (200 mg/kg body weight administered in 3 daily doses by oral gavage) to induce CreERT2-mediated deletion of the floxed Bcl-x alleles. Mice were monitored for up to 200 days post-treatment with tamoxifen. Data are presented as % survival post-treatment with tamoxifen, and statistical significance was assessed using the Mantel–Cox (log-rank) test; ****P < 0.0001. B. Bcl-xfl/fl;RosaCreERT2+/Ki (n = 24) or, as controls, Bcl-xfl/fl (n = 9), and RosaCreERT2+/Ki (n = 6) mice (males and females, aged 8–14 weeks, numbers also indicated in the figure) were lethally γ-irradiated (2 × 5.5 Gy, 3 h apart) and reconstituted with bone marrow from UBC-GFP mice (referred to as GFP-Chimeras). After 8 weeks, reconstituted mice were treated with tamoxifen (200 mg/kg body weight administered in 3 daily doses oral gavage) and monitored for up to 200 days (termination of the experiment). Data are presented as % survival post-treatment with tamoxifen, and statistical significance was assessed using the Mantel–Cox (log-rank) test; ****P < 0.0001. C–F. Total counts of (C) platelets, (D) red blood cells (RBC), (E) hemoglobin (HGB) content, and (F) hematocrit (HCT) of tamoxifen-treated Bcl-xfl/fl;RosaCreERT2+/Ki (n = 8) or, as controls, Bcl-xfl/fl (n = 6) and RosaCreERT2+/Ki (n = 12) and Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimera (n = 21) or, as controls, Bcl-xfl/fl;GFP-Chimera (n = 6) and RosaCreERT2+/Ki;GFP-Chimera (n = 6) mice were determined by ADVIA. Data are presented as mean ± SEM. Each data point represents an individual mouse. Statistical significance was assessed using Student's t-test; ****P < 0.0001. G. Spleen weights were measured in sick mice (at sacrifice) or, for the healthy control mice, at the termination of the experiment. Data are presented as mean ± SEM. Each data point represents an individual mouse and numbers are indicated. Statistical significance was assessed using Student's t-test; ****P < 0.0001. H. Representative image of enlarged spleens from two Bcl-xfl/fl;RosaCreERT2Ki/+ mice and age-matched control Bcl-xfl/fl and RosaCreERT2+/Ki mice (34 days after treatment). I. Histological analysis of H&E-stained sections of spleens of tamoxifen-treated mice of the indicated genotypes. Download figure Download PowerPoint To identify the essential functions of BCL-XL in non-hematopoietic tissues of adult mice, we generated bone marrow chimeras: Bcl-xfl/fl;RosaCreERT2+/Ki mice that had been lethally irradiated and reconstituted with bone marrow cells from UBC-GFP mice (GFP-Chimeras; experimental design in Appendix Fig S1B). Unexpectedly, these chimeric animals also presented with severe anemia and thrombocytopenia after loss of BCL-XL, albeit at a considerably later time (Fig 1C–F). Around day 100 post-treatment with tamoxifen, the Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras appeared runty, lethargic, and anemic and had to be sacrificed according to our animal ethics guidelines after > 15% body weight loss (median survival = 102 days, Fig 1B). Notably, sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras did not present with splenomegaly (Fig 1G) or an increase in the red pulp area of the spleen (Fig 1I), which are hallmarks of primary anemia caused by loss of BCL-XL in erythroid and megakaryocytic cell populations (Fig 1G–I). Importantly, the tamoxifen-treated Bcl-xfl/fl;GFP-Chimera and RosaCreERT2+/Ki;GFP-Chimera control mice all survived without any adverse events for > 240 days (termination of the experiment). These findings demonstrate that inducible loss of BCL-XL in all tissues of adult mice (including hematopoietic cells) causes primary anemia and thrombocytopenia, whereas the combination of total body γ-irradiation (TBI), followed by rescue with a UBC-GFP hematopoietic system, and inducible loss of BCL-XL only in non-hematopoietic cells causes secondary anemia. The secondary anemia in mice caused by the combination of γ-radiation and inducible deletion of BCL-XL is not driven by inflammation, hematopoietic malignancy, or liver damage TBI is commonly used for the treatment of hematologic malignancies, often alongside high dose chemotherapy, prior to hematopoietic stem/progenitor cell (HSPC) transplantation. TBI causes many side-effects, including nausea, diarrhea, sensitive skin, and hair loss, that are temporary. However, TBI can also cause severe long-term damage to several organs, such as the lung, trachea, and mouth (radiation-induced pneumonitis, fibrosis, oral mucositis) (Marks et al, 2003; Mehta, 2005), reproductive system (radiation-induced infertility) (Ogilvy-Stuart & Shalet, 1993), liver (radiation-induced liver disease) (Kim & Jung, 2017), gastrointestinal tract (radiation-induced gastric mucositis, radiation-induced gastrointestinal syndrome) (Francois et al, 2013; Olcina & Giaccia, 2016), or kidney (radiation-induced nephropathy) (Cohen, 2000; Cohen & Robbins, 2003). Furthermore, TBI can also initiate secondary malignancies (Dracham et al, 2018). The above-mentioned TBI-induced pathologies frequently cause secondary anemia (Weiss & Goodnough, 2005; Davis & Littlewood, 2012). For example, hematologic malignancies impair the production of RBCs in the bone marrow as a consequence of competing for essential growth factors, the production of reactive oxygen species (ROS) and pro-inflammatory cytokines that damage erythroid progenitors (Weiss & Goodnough, 2005; Davis & Littlewood, 2012). Moreover, a robust inflammatory response is observed upon radiation-induced damage to the gastrointestinal tract (Olcina & Giaccia, 2016). Pro-inflammatory cytokines (e.g., IL-6, TNF-α) interfere with the production of erythropoietin (EPO) and the availability of iron, both crucial for RBC development. Furthermore, inflammatory cytokines enhance the production of white blood cells (WBCs) and can thereby decrease the differentiation of progenitors into RBCs (Weiss & Goodnough, 2005). We did not detect signs of ongoing inflammation in the tamoxifen-treated Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras as shown by the observation that the numbers of WBCs (Fig 2A), including lymphocytes, neutrophils, basophils, eosinophils, and monocytes, were all within the normal range (Appendix Fig S2A). The neutrophil-to-lymphocyte ratio (NLR), even though increased in the tamoxifen-treated Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras, was still within the normal range (Fig 2B). Moreover, no signs of hematologic malignancies, such as increased numbers of leukocytes, were observed in the blood or bone marrow of the tamoxifen-treated Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (Fig 2A and C). Figure 2. The combination of γ-radiation and inducible deletion of BCL-XL causes neither inflammation, hematopoietic malignancy, nor liver damage Bcl-xfl/fl;RosaCreERT2+/Ki (n = 8) or, as controls, Bcl-xfl/fl (n = 6), and RosaCreERT2+/Ki (n = 12) mice as well as Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n = 21), or as controls, Bcl-xfl/fl;GFP-Chimeras (n = 6), and RosaCreERT2+/Ki;GFP-Chimeras (n = 6) (age 8–14 weeks, males and females) were treated with tamoxifen (200 mg/kg body weight administered in 3 daily doses by oral gavage) to induce CreERT2-mediated deletion of the floxed Bcl-x alleles. Total white blood cell counts (WBC) were analyzed by ADVIA in sick mice or at the termination of the experiment (healthy control mice). Data are presented as mean ± SEM. Each data point represents an individual mouse and n numbers are indicated above. Statistical significance was assessed using Student's t-test. No statistically significant differences were observed. The neutrophil/lymphocyte ratio (NLR) is presented as mean ± SEM for sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n = 21) or healthy control RosaCreERT2+/Ki;GFP-Chimeras (n = 6) at the termination of the experiment. Data are presented as mean ± SEM. Each data point represents an individual mouse. Statistical significance was assessed using Student's t-test; *P < 0.05. Histological analysis of H&E-stained sections of the sternum of sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras or healthy wild-type and RosaCreERT2+/Ki;GFP-Chimera control mice at the indicated time points post-treatment with tamoxifen (dpt = days post-treatment). Histological analysis of H&E-stained sections of the livers of sick mice or age-matched healthy control wild-type mice or healthy RosaCreERT2+/Ki;GFP-Chimeras at the termination of the experiment (dpt = days post-treatment). ALT (left panel), AST (middle panel), and bilirubin levels (right panel) in the serum were determined in sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n = 18) or in healthy RosaCreERT2+/Ki;GFP-Chimeras (n = 4) at the termination of the experiment. Data are presented as mean ± SEM. Each data point represents one individual mouse. Statistical significance was assessed using Student's t-test; **P < 0.01. nd = not detected. Download figure Download PowerPoint The liver represents the primary storage site of iron and source of hepcidin that are both essential for RBC production. Hence, secondary anemia can result from liver damage. Histological analysis failed to reveal any abnormalities in the livers of the tamoxifen-treated Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (Fig 2D), and no marked increases in the liver enzyme ALT or unconjugated bilirubin were detected in their sera (Fig 2E). However, these animals presented with increased levels of AST (Fig 2E), but unlike ALT, AST is also found in the heart, skeletal muscle, kidney, and RBCs. Thus, with the lack of other indicators of liver pathology, we attributed this AST increase to non-liver toxicity. These findings demonstrate that the secondary anemia in the tamoxifen-treated Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras is not a consequence of erythroid cell destruction, hematologic malignancy, chronic infection, inflammation, nor liver damage. Instead, these results suggest that the secondary anemia might be caused by radiation-induced kidney damage. The combination of γ-radiation and inducible deletion of BCL-XL causes severe kidney damage in adult mice EPO stimulates erythropoiesis and is produced in response to cellular hypoxia in the kidney by interstitial fibroblasts adjacent to the renal proximal convoluted tubule and peritubular capillaries. Acute and chronic kidney disease that damages these cells decreases the production of EPO, thereby compromising the generation of erythroid progenitors (CFU-e) and thus RBC production. We hypothesized that the secondary anemia observed in tamoxifen-treated Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras results from damage to the EPO-secreting cells in the kidney. Accordingly, urine tests showed severe proteinuria in sick tamoxifen-treated Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (Appendix Fig S3A), and due to increased urination, the bedding of their cages had to be changed frequently. The kidneys of these sick mice were abnormally small and yellow (Fig 3A and B). Histological analysis revealed severe renal tubulointerstitial disease with segmental areas showing diminution of the renal tubules, interstitial fibrosis, and patchy chronic inflammation with focal and segmental glomerular changes and thickening of renal arterioles. The renal medulla and papillae contained intraluminal calcified cellular debris and crystalline precipitates within the collecting ducts and dilated loops of Henle, where urine is concentrated (Fig 3C). This was sometimes accompanied by intraluminal multi-nucleated macrophages, reminiscent of the changes seen in myeloma nephropathy. The glomeruli in the affected segments showed secondary focal and segmental sclerosis. These structural changes were additionally visualized using confocal microscopy (Appendix Fig S4). Figure 3. The combination of γ-radiation and inducible deletion of BCL-XL causes severe kidney damage Bcl-xfl/fl;RosaCreERT2+/Ki (n = 7), or as controls, Bcl-xfl/fl (n = 8), and RosaCreERT2+/Ki (n = 16) mice as well as Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n = 5), Bcl-xfl/fl;GFP-Chimeras (n = 8), and RosaCreERT2+/Ki;GFP-Chimeras (n = 6) (age 8–14 weeks, m
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