Loss of endogenous thymosin β4 accelerates glomerular disease
Authors: Elisavet Vasilopoulou, Maria Kolatsi-Joannou, Maja T. Lindenmeyer, Kathryn E. White, Michael G. Robson, Clemens D. Cohen, Neil J. Sebire, Paul R. Riley, Paul J. Winyard, David A. Long
Journal: Kidney International (2016)

Glomerular disease is characterized by morphologic changes in podocyte cells accompanied by inflammation and fibrosis. Thymosin β4 regulates cell morphology, inflammation, and fibrosis in several organs and administration of exogenous thymosin β4 improves animal models of unilateral ureteral obstruction and diabetic nephropathy. However, the role of endogenous thymosin β4 in the kidney is unknown. We demonstrate that thymosin β4 is expressed prominently in podocytes of developing and adult mouse glomeruli. Global loss of thymosin β4 did not affect healthy glomeruli, but accelerated the severity of immune-mediated nephrotoxic nephritis with worse renal function, periglomerular inflammation, and fibrosis. Lack of thymosin β4 in nephrotoxic nephritis led to the redistribution of podocytes from the glomerular tuft toward the Bowman capsule suggesting a role for thymosin β4 in the migration of these cells. Thymosin β4 knockdown in cultured podocytes also increased migration in a wound-healing assay, accompanied by F-actin rearrangement and increased RhoA activity. We propose that endogenous thymosin β4 is a modifier of glomerular injury, likely having a protective role acting as a brake to slow disease progression. Glomerular disease is characterized by morphologic changes in podocyte cells accompanied by inflammation and fibrosis. Thymosin β4 regulates cell morphology, inflammation, and fibrosis in several organs and administration of exogenous thymosin β4 improves animal models of unilateral ureteral obstruction and diabetic nephropathy. However, the role of endogenous thymosin β4 in the kidney is unknown. We demonstrate that thymosin β4 is expressed prominently in podocytes of developing and adult mouse glomeruli. Global loss of thymosin β4 did not affect healthy glomeruli, but accelerated the severity of immune-mediated nephrotoxic nephritis with worse renal function, periglomerular inflammation, and fibrosis. Lack of thymosin β4 in nephrotoxic nephritis led to the redistribution of podocytes from the glomerular tuft toward the Bowman capsule suggesting a role for thymosin β4 in the migration of these cells. Thymosin β4 knockdown in cultured podocytes also increased migration in a wound-healing assay, accompanied by F-actin rearrangement and increased RhoA activity. We propose that endogenous thymosin β4 is a modifier of glomerular injury, likely having a protective role acting as a brake to slow disease progression. End-stage renal failure is a devastating condition requiring lifelong dialysis and transplantation and a risk factor for all-cause mortality and cardiovascular disease.1Eckardt K.U. Coresh J. Devuyst O. et al.Evolving importance of kidney disease: from subspecialty to global health burden.Lancet. 2013; 382: 158-169Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar Many cases are due to disruption of the glomerular filtration barrier, consisting of epithelial podocytes, the endothelium, the mesangium, and the glomerular basement membrane.2Greka A. Mundel P. Cell biology and pathology of podocytes.Annu Rev Physiol. 2012; 74: 299-323Crossref PubMed Scopus (376) Google Scholar Podocytes have a unique shape maintained by a complex cytoskeleton,3Welsh G.I. Saleem M.A. The podocyte cytoskeleton–key to a functioning glomerulus in health and disease.Nat Rev Nephrol. 2012; 8: 14-21Crossref Scopus (196) Google Scholar with branched foot process extensions that abut each other at slit diaphragms. During glomerular injury, the podocyte architecture is perturbed, resulting in defective filtration and proteinuria,2Greka A. Mundel P. 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Gili E. et al.Thymosin beta4 protects C57BL/6 mice from bleomycin-induced damage in the lung.Eur J Clin Invest. 2013; 43: 309-315Crossref Scopus (26) Google Scholar Recent studies investigated exogenous Tmsb4x as a treatment for kidney disease.17Vasilopoulou E. Winyard P.J. Riley P.R. et al.The role of thymosin-beta4 in kidney disease.Expert Opin Biol Ther. 2015; 15: S187-S190Crossref Scopus (12) Google Scholar Tmsb4x reduced renal tubulointerstitial fibrosis after unilateral ureteral obstruction in mice, potentially through decreasing plasminogen activator inhibitor-1 expression and dampening transforming growth factor-β1 signaling.18Zuo Y. Chun B. Potthoff S.A. et al.Thymosin beta4 and its degradation product, Ac-SDKP, are novel reparative factors in renal fibrosis.Kidney Int. 2013; 84: 1166-1175Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar In KK Cg-Ay/J mice, a model of type 2 diabetes mellitus, daily Tmsb4x treatment for 3 months reduced albuminuria and attenuated renal pathology.19Zhu J. Su L.P. Zhou Y. et al.Thymosin beta4 attenuates early diabetic nephropathy in a mouse model of type 2 diabetes mellitus.Am J Ther. 2015; 22: 141-146Crossref Scopus (13) Google Scholar Furthermore, N-acetyl-seryl-aspartyl-lysyl-proline, the N-terminal tetrapeptide generated by Tmsb4x cleavage,20Cavasin M.A. Rhaleb N.E. Yang X.P. et al.Prolyl oligopeptidase is involved in release of the antifibrotic peptide Ac-SDKP.Hypertension. 2004; 43: 1140-1145Crossref PubMed Scopus (111) Google Scholar has beneficial effects on fibrosis and inflammation in unilateral ureteral obstruction, remnant kidneys, diabetic nephropathy, and glomerulonephritis.18Zuo Y. Chun B. Potthoff S.A. et al.Thymosin beta4 and its degradation product, Ac-SDKP, are novel reparative factors in renal fibrosis.Kidney Int. 2013; 84: 1166-1175Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 21Liao T.D. Yang X.P. D'Ambrosio M. et al.N-acetyl-seryl-aspartyl-lysyl-proline attenuates renal injury and dysfunction in hypertensive rats with reduced renal mass: council for high blood pressure research.Hypertension. 2010; 55: 459-467Crossref PubMed Scopus (44) Google Scholar, 22Omata M. Taniguchi H. Koya D. et al.N-acetyl-seryl-aspartyl-lysyl-proline ameliorates the progression of renal dysfunction and fibrosis in WKY rats with established anti-glomerular basement membrane nephritis.J Am Soc Nephrol. 2006; 17: 674-685Crossref PubMed Scopus (50) Google Scholar, 23Rhaleb N.E. Pokharel S. Sharma U. et al.Renal protective effects of N-acetyl-Ser-Asp-Lys-Pro in deoxycorticosterone acetate-salt hypertensive mice.J Hypertension. 2011; 29: 330-338Crossref PubMed Scopus (34) Google Scholar Tmsb4x transcripts are detectable by in situ hybridization in developing and adult glomeruli,24Guinobert I. Viltard M. Piquemal D. et al.Identification of differentially expressed genes between fetal and adult mouse kidney: candidate gene in kidney development.Nephron Physiol. 2006; 102: 81-91Crossref Scopus (9) Google Scholar with strong expression in podocytes.24Guinobert I. Viltard M. Piquemal D. et al.Identification of differentially expressed genes between fetal and adult mouse kidney: candidate gene in kidney development.Nephron Physiol. 2006; 102: 81-91Crossref Scopus (9) Google Scholar, 25Brunskill E.W. Georgas K. Rumballe B. et al.Defining the molecular character of the developing and adult kidney podocyte.PLoS One. 2011; 6: e24640Crossref PubMed Scopus (103) Google Scholar Furthermore, in rat remnant kidneys, proteomic analysis of laser-capture dissected glomeruli demonstrated significantly increased Tmsb4x in sclerotic versus normal glomeruli.26Xu B.J. Shyr Y. Liang X. et al.Proteomic patterns and prediction of glomerulosclerosis and its mechanisms.J Am Soc Nephrol. 2005; 16: 2967-2975Crossref PubMed Scopus (87) Google Scholar Despite such evidence of expression and beneficial renal effects, the functional importance of endogenous Tmsb4x in the kidney during health and disease is completely unknown. In this study, we confirmed that Tmsb4x is highly expressed in the kidney glomerulus, predominantly in podocytes. Using global Tmsb4x knockout mice,27Rossdeutsch A. Smart N. Dube K.N. et al.Essential role for thymosin beta4 in regulating vascular smooth muscle cell development and vessel wall stability.Circ Res. 2012; 111: e89-e102Crossref Scopus (44) Google Scholar we demonstrated that endogenous Tmsb4x was dispensable in healthy glomeruli. Furthermore, in an experimental model of glomerular damage, lack of Tmsb4x worsened disease progression by (i) enhancing podocyte migration, facilitating their redistribution from the glomerular tuft to the Bowman capsule, and (ii) increasing periglomerular inflammation and interstitial fibrosis. Thus, we provide the first evidence that endogenous Tmsb4x is critical in the progression of glomerular disease. Tmsb4x mRNA levels were assessed in the spleen, liver, heart, and whole kidney of healthy adult mice. The highest transcript levels were found in the spleen with ∼10 times less Tmsb4x in the kidney (Figure 1a). Using Dynabead perfusion (Thermo Fisher Scientific, Waltham, MA),28Long D.A. Kolatsi-Joannou M. Price K.L. et al.Albuminuria is associated with too few glomeruli and too much testosterone.Kidney Int. 2013; 83: 1118-1129Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar we isolated glomeruli and found that Tmsb4x levels were significantly enriched in glomeruli compared with the rest of the kidney (Figure 1a). In situ hybridization detected Tmsb4x expression in immature glomeruli of embryonic day 16.5 developing kidneys, predominantly in podocytes (Figure 1b and c). The protein expression of Tmsb4x was also assessed by immunohistochemistry, and we found strong localization in glomerular podocytes at embryonic day 18 (Figure 1d). This expression pattern was maintained in 1-week-old postnatal (Figure 1e) and 8-week-old adult kidneys (Figure 1f). Tmsb4x podocyte expression was further confirmed by colocalization of Tmsb4x with nephrin (Nphs1), a slit diaphragm component29Li X. Chuang P.Y. D'Agati V.D. et al.Nephrin preserves podocyte viability and glomerular structure and function in adult kidneys.J Am Soc Nephrol. 2015; 26: 2361-2377Crossref Scopus (77) Google Scholar (Figure 1g–i), and nestin, an intermediate filament protein expressed in mature podocytes30Thorner P.S. Ho M. Eremina V. et al.Podocytes contribute to the formation of glomerular crescents.J Am Soc Nephrol. 2008; 19: 495-502Crossref PubMed Scopus (107) Google Scholar (Supplementary Figure S1A–C). In contrast, Tmsb4x did not colocalize with the pan-endothelial marker Cd31 (Supplementary Figure S1D–F). We examined mice with a global loss of Tmsb4x27Rossdeutsch A. Smart N. Dube K.N. et al.Essential role for thymosin beta4 in regulating vascular smooth muscle cell development and vessel wall stability.Circ Res. 2012; 111: e89-e102Crossref Scopus (44) Google Scholar to assess the importance of endogenous Tmsb4x in healthy glomeruli. As Tmsb4x is mapped to the X chromosome,31Li X. Zimmerman A. Copeland N.G. et al.The mouse thymosin beta 4 gene: structure, promoter identification, and chromosome localization.Genomics. 1996; 32: 388-394Crossref PubMed Scopus (28) Google Scholar we crossed hemizygous null male mice (Tmsb4x−/y) with heterozygous Tmsb4x+/− adult female mice (Figure 2a). We found no lethal developmental abnormalities with the offspring conforming to Mendelian ratios (Supplementary Table S1). Tmsb4x−/y mice had albumin excretion (Figure 2b) and blood urea nitrogen (Figure 2c) levels similar to those of male wild-type Tmsb4x+/y mice at the ages of 1, 3, and 6 months. There was no difference in body weight at 1 (Tmsb4x+/y = 23.8 ± 0.7 g; Tmsb4x−/y = 23.4 ± 0.4 g), 3 (Tmsb4x+/y = 30.0 ± 0.5 g; Tmsb4x−/y = 29.2 ± 0.7 g), or 6 (Tmsb4x+/y = 35.8 ± 1.0 g; Tmsb4x−/y = 35.3 ± 1.6 g) months of age. Using semiquantitative analysis of light microscopy images, we found no differences in glomerular morphology between 6-month-old Tmsb4x+/y and Tmsb4x−/y mice (Figure 2d–f). This was confirmed by transmission electron microscopy with normal foot process architecture, laminar structure of the basement membrane, and the presence of endothelial fenestrae in both Tmsb4x+/y and Tmsb4x−/y mice (Figure 2g and h). We demonstrated the loss of Tmsb4x protein in glomeruli of Tmsb4x−/y mice compared with Tmsb4x+/y (Figure 2i and j), along with undetectable Tmsb4x mRNA levels in whole kidneys (Figure 2k). We examined whether there was any compensation for the lack of Tmsb4x by other β-thymosins, but found no changes in the renal mRNA levels of Tmsb10, Tmsb15a, Tmsb15b, and Tmsb15l (Figure 2l–o). We also assessed mRNA levels of genes involved in actin polymerization and found no differences in profilin (Pfn) 1 and 2 and destrin (Dstn) between Tmsb4x−/y and Tmsb4x+/y mice (Supplementary Figure S2A–C). In contrast, cofilin 1 (Cfl1) mRNA levels were significantly increased in Tmsb4x−/y kidneys by ∼30% compared with Tmsb4x+/y mice (Supplementary Figure S2D). We specifically examined Cfl1 mRNA levels in podocytes and found no change after knock down of endogenous Tmsb4x by small, interfering RNA (siRNA) (Supplementary Figure S2E). Furthermore, there was no difference in Cfl1 mRNA levels in primary podocytes isolated from Tmsb4x−/y and Tmsb4x+/y mice (Supplementary Figure S2F). Finally, the mRNA levels of genes important for podocyte function (Nphs1, Nphs2, Synpo, Cd2ap, and Wt1) were unchanged in Tmsb4x−/y and Tmsb4x+/y kidneys (Supplementary Figure S3A–E). Our results suggest that the lack of Tmsb4x does not affect the function and morphology of healthy glomeruli. Therefore, we investigated whether Tmsb4x has a role in glomerular disease. We utilized the nephrotoxic serum (NTS) nephritis model, which replicates some of the pathologic features of human crescentic glomerulonephritis.32Pippin J.W. Brinkkoetter P.T. Cormack-Aboud F.C. et al.Inducible rodent models of acquired podocyte diseases.Am J Physiol Renal Physiol. 2009; 296: F213-F229Crossref PubMed Scopus (211) Google Scholar NTS nephritis involves the injury of intrinsic glomerular cells, including podocytes, as well as leukocyte infiltration, glomerulosclerosis, and tubulointerstitial fibrosis,33Khan S.B. Cook H.T. Bhangal G. et al.Antibody blockade of TNF-alpha reduces inflammation and scarring in experimental crescentic glomerulonephritis.Kidney Int. 2005; 67: 1812-1820Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar processes in which Tmsb4x has been implicated.10Conte E. Genovese T. Gili E. et al.Thymosin beta4 protects C57BL/6 mice from bleomycin-induced damage in the lung.Eur J Clin Invest. 2013; 43: 309-315Crossref Scopus (26) Google Scholar, 16Sosne G. Qiu P. Christopherson P.L. et al.Thymosin beta 4 suppression of corneal NFkappaB: a potential anti-inflammatory pathway.Exp Eye Res. 2007; 84: 663-669Crossref PubMed Scopus (131) Google Scholar We predicted that the lack of global Tmsb4x may exacerbate NTS nephritis severity and examined this in 3-month-old Tmsb4x−/y and Tmsb4x+/y mice (Figure 3a). Albuminuria and the albumin/creatinine ratio were significantly increased in Tmsb4x+/y mice 21 days after NTS administration compared with the levels before immunization (P < 0.001 in both cases) (Figure 3b and c). Strikingly, both albuminuria and the albumin/creatinine ratio were further enhanced by ∼5- and 7-fold, respectively, when NTS was injected in Tmsb4x−/y mice compared with Tmsb4x+/y mice (P < 0.01 in both cases) (Figure 3b and c). Administration of NTS to Tmsb4x−/y mice also significantly increased plasma creatinine (P < 0.01) (Figure 3d), impaired creatinine clearance (P < 0.01) (Figure 3e), and raised the blood urea nitrogen level (P < 0.05) (Figure 3f) compared with Tmsb4x+/y mice with NTS nephritis. We found no difference in Tmsb4x levels in whole kidneys obtained from Tmsb4x+/y mice without disease or administered NTS (Supplementary Figure S4A). Furthermore, in kidney biopsy specimens obtained from patients with either rapidly progressive glomerulonephritis or lupus nephritis, there was no change in glomerular or tubulointerstitial TMSB4X mRNA levels compared with living donor control kidneys (Supplementary Figure S4B and C). Seven days after NTS administration, we observed mild glomerular injury in Tmsb4x−/y and Tmsb4x+/y mice, with some glomeruli containing hyaline deposits, increased mesangial matrix, and occasional adhesion of the glomerular tuft to the Bowman capsule (Supplementary Figure S5A–C). After 21 days, there was a range of abnormalities in Tmsb4x−/y and Tmsb4x+/y mice injected with NTS including collapse of capillary loops, segmental or global glomerulosclerosis, adhesion of the glomerular tuft to the Bowman capsule, and glomerular epithelial hyperplasia lesions, a feature of early crescent formation in this model.34Le Hir M. Keller C. Eschmann V. et al.Podocyte bridges between the tuft and Bowman's capsule: an early event in experimental crescentic glomerulonephritis.J Am Soc Nephrol. 2001; 12: 2060-2071PubMed Google Scholar Semiquantitative histologic scoring (Supplementary Figure S6A–E) by 2 blinded observers revealed that Tmsb4x+/y mice injected with NTS had a significantly increased mean glomerular score compared with Tmsb4x+/y mice without disease (P < 0.001) (Figure 3g–j). Glomeruli of Tmsb4x−/y administered NTS had an even higher glomerular mean score that was significantly greater than that in Tmsb4x+/y mice with NTS nephritis (P < 0.05). This was associated with an increased proportion of sclerotic glomeruli and an increased incidence of epithelial hyperplasia lesions in Tmsb4x−/y compared with Tmsb4x+/y mice administered NTS (Supplementary Figure S6F). We examined whether the difference in disease severity between Tmsb4x−/y and Tmsb4x+/y mice administered NTS could be due to a decrease in binding of the antiglomerular antibody but found no difference in the amount of sheep IgG deposited within the glomerulus (Supplementary Figure S7A–C). We assessed whether the lack of Tmsb4x changes the humoral immune response to sheep IgG. NTS injection led to significantly increased production of circulating murine IgG1 and IgG2a, but not IgG2b or IgG3, against sheep IgG compared with Tmsb4x+/y mice without disease. However, there was no difference in the plasma titers of any of the IgG subclasses between Tmsb4x−/y and Tmsb4x+/y mice administered NTS (Supplementary Figure S7D–G). After NTS nephritis, we found that Tmsb4x still colocalized with Nphs1 (Figure 4a–c) and subsequently examined the effect that a lack of Tmsb4x had on podocytes in this model. First, we quantified Wilms Tumor 1 postive (WT1+) podocyte numbers,35Guo J.K. Menke A.L. Gubler M.C. et al.WT1 is a key regulator of podocyte function: reduced expression levels cause crescentic glomerulonephritis and mesangial sclerosis.Human Mol Genet. 2002; 11: 651-659Crossref PubMed Scopus (221) Google Scholar inside and outside of the glomerular tuft (Figure 4d–f). The number of glomerular tuft WT1+ cells was unchanged in Tmsb4x+/y mice after NTS injection compared with mice without disease. However, NTS administration to Tmsb4x−/y mice significantly reduced the number of WT1+ glomerular tuft cells compared with Tmsb4x+/y mice with NTS nephritis (P < 0.001) (Figure 4g). This finding persisted after normalizing the WT1+ cell number to the glomerular tuft area (P < 0.05) (Figure 4h). In contrast, there was an increased number of WT1+ cells outside the glomerular tuft in both Tmsb4x+/y and Tmsb4x−/y mice after NTS administration; this was more prominent and significantly different in the Tmsb4x−/y animals (P < 0.05 compared with Tmsb4x+/y without disease) (Figure 4i). The total number of podocytes in the whole glomerulus did not differ between any of the groups (Figure 4j), suggesting that lack of Tmsb4x leads to podocyte redistribution from the glomerular tuft toward the Bowman capsule rather than affecting podocyte cell death. To support this, we assessed podocyte apoptosis using terminal deoxynucleotidyltransferase–mediated dUTP nick end-labeling (TUNEL)36Davis B. Dei Cas A. Long D.A. et al.Podocyte-specific expression of angiopoietin-2 causes proteinuria and apoptosis of glomerular endothelia.J Am Soc Nephrol. 2007; 18: 2320-2329Crossref PubMed Scopus (123) Google Scholar in combination with WT1 staining (Supplementary Figure S8A–C). Administration of NTS increased the number of glomerular apoptotic cells compared with healthy mice, and this effect was significant in Tmsb4+/y mice with glomerular disease (P < 0.05) (Supplementary Figure S8D). However, the number of TUNEL+/WT1+ cells was not significantly different between any of the groups (Supplementary Figure S8E). We postulated that the redistribution of podocytes in nephrotoxic nephritis may be due to changes in cell migration driven by lack of Tmsb4x. Therefore, we transfected cultured differentiated mouse podocytes37Mundel P. Reiser J. Zuniga Mejia Borja A. et al.Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines.Exp Cell Res. 1997; 236: 248-258Crossref PubMed Scopus (765) Google Scholar with siRNA against Tmsb4x (Figure 5a); this resulted in >90% knockdown in Tmsb4x levels (Figure 5b) (P < 0.001). Knockdown of endogenous Tmsb4x did not affect podocyte viability (Figure 5c) but increased the number of cells that migrated into the wound area in a wound-healing assay (P < 0.05) (Figure 5d and e). Because the cytoskeleton is essential for cell movement,38Pollard T.D. Cooper J.A. Actin, a central player in cell shape and movement.Science. 2009; 326: 1208-1212Crossref PubMed Scopus (1336) Google Scholar we visualized podocyte actin by phalloidin staining and classified the filament organization as either cytoplasmic stress fibers (Figure 5f) or cortical actin (Figure 5g). Knockdown of endogenous Tmsb4x significantly increased the percentage of cells with stress actin fiber organization (P < 0.001) (Figure 5h). Finally, we assessed the effects of Tmsb4x knockdown on the activation of RhoA and Cdc42, which regulate actin dynamics and cell migration.39Raftopoulou M. Hall A. Cell migration: Rho GTPases lead the way.Dev Biol. 2004; 265: 23-32Crossref PubMed Scopus (1142) Google Scholar There was increased RhoA activity in podocytes transfected with Tmsb4x siRNA compared with control siRNA (P < 0.05) (Figure 5i), whereas Cdc42 activity was unaffected (Figure 5j). Tmsb4x is expressed in macrophages18Zuo Y. Chun B. Potthoff S.A. et al.Thymosin beta4 and its degradation product, Ac-SDKP, are novel reparative factors in renal fibrosis.Kidney Int. 2013; 84: 1166-1175Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 40Hannappel E. Xu G.J. Morgan J. et al.Thymosin beta 4: a ubiquitous peptide in rat and mouse tissues.Proc Natl Acad Sci U S A. 1982; 79: 2172-2175Crossref PubMed Scopus (148) Google Scholar and reduces inflammation in several disease settings.10Conte E. Genovese T. Gili E. et al.Thymosin beta4 protects C57BL/6 mice from bleomycin-induced damage in the lung.Eur J Clin Invest. 2013; 43: 309-315Crossref Scopus (26) Google Scholar, 16Sosne G. Qiu P. Christopherson P.L. et al.Thymosin beta 4 suppression of corneal NFkappaB: a potential anti-inflammatory pathway.Exp Eye Res. 2007; 84: 663-669Crossref PubMed Scopus (131) Google Scholar, 18Zuo Y. Chun B. Potthoff S.A. et al.Thymosin beta4 and its degradation product, Ac-SDKP, are novel reparative factors in renal fibrosis.Kidney Int. 2013; 84: 1166-1175Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 41Peng H. Xu J. Yang X.P. et al.Thymosin-beta4 prevents cardiac rupture and improves cardiac function in mice with myocardial infarction.Am J Physiol Heart Circ Physiol. 2014; 307: H741-H751Crossref PubMed Scopus (28) Google Scholar As immune cell infiltration plays a critical role in the initiation and progression of crescentic glomerulonephritis,42Duffield J.S. Tipping P.G. Kipari T. et al.Conditional ablation of macrophages halts progression of crescentic glomerulonephritis.Am J Pathol. 2005; 167: 1207-1219Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 43Huang X.R. Tipping P.G. Apostolopoulos J. et al.Mechanisms of T cell-induced glomerular injury in anti-glomerular basement membrane (GBM) glomerulonephritis in rats.Clin Exp Immunol. 1997; 109: 134-142Crossref PubMed Scopus (97) Google Scholar, 44Tipping P.G. Huang X.R. Qi M. et al.Crescentic glomerulonephritis in CD4- and CD8-deficient mice. Requirement for CD4 but not CD8 cells.Am J Pathol. 1998; 152: 1541-1548PubMed Google Scholar we decided to examine this in the NTS nephritis model. We found expression of Tmsb4x in F4/80+ macrophages surrounding the glomeruli and occasionally within the glomerular tuft (Figure 6a–c) and went on to examine the effect of Tmsb4x loss on glomerular inflammation in our experimental model. We measured the number of Cd3+ (T cells) and F4/80+ cells in Tmsb4x−/y and Tmsb4x+/y glomeruli. Twenty-one days after NTS injection, there was a significant increase in Cd3+ cells in the glomerular tuft of Tmsb4x−/y mice compared with Tmsb4x+/y mice without disease (P < 0.05) but no significant difference when comparing Tmsb4x−/y and Tmsb4x+/y mice with nephrotoxic nephritis (Figure 6d–g). There was also no difference in the number of periglomerular Cd3+ cells between experimental groups (Figure 6h). Seven days after NTS administration, the number of F4/80+ glomerular tuft cells was similar in all experimental groups, but were significantly increased in the periglomerular area of both NTS-injected Tmsb4x+/y and Tmsb4x−/y mice compared with healthy mice (P < 0.01) (Supplementary Figure S9A and B). The accumulation of F4/80+ cells persisted in Tmsb4x−/y mice 21 days after NTS administration, with increased numbers in both the glomerular tuft and periglomerular area compared with Tmsb4x+/y mice with or without disease (P < 0.01) (Figure 6i–m). mRNA levels of the pan-macrophage marker Cd68 were also significantly higher 21 days after NTS injection in whole-kidney homogenates obtained from Tmsb4x−/y compared with Tmsb4x+/y mice (Supplementary Figure S10A). Cd68 is expressed by all macrophages, but these comprise a diverse group that includes a broad spectrum of cellular phenotypes, often characterized as pro-inflammatory (M1 type) and tissue repair (M2 type) macrophages. We quantified the mRNA levels of M1 (Mcp1, Cd86) and M2 markers (Cd206, Arg1) and found all of these genes were