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• W2901029372 abstract "Bone morphogenetic protein (BMP) signaling has been shown to modulate the development of renal fibrosis in animal models of kidney injury, but the downstream mediators are incompletely understood. In wild-type mice, canonical BMP signaling mediated by SMAD1/5/8 transcription factors was constitutively active in healthy renal tubules, transiently down-regulated after ischemia reperfusion injury (IRI), and reactivated during successful tubular regeneration. We then induced IRI in mice with a tubular-specific BMP receptor 1A (BMPR1A) deletion. These mice failed to reactivate SMAD1/5/8 signaling in the post-ischemic phase and developed renal fibrosis after injury. Using unbiased genomic analyses, we identified three genes encoding inhibitor of DNA-binding (ID) proteins (Id1, Id2, and Id4) as key targets of BMPR1A-SMAD1/5/8 signaling. BMPR1A-deficient mice failed to re-induce these targets following IRI. Instead, BMPR1A-deficiency resulted in activation of pro-fibrotic signaling proteins that are normally repressed by ID proteins, namely, p38 mitogen-activated protein kinase and cell cycle inhibitor p27. These data indicate that the post-ischemic activation of canonical BMP signaling acts endogenously to repress pro-fibrotic signaling in tubular cells and may help to prevent the progression of acute kidney injury to chronic kidney disease. Bone morphogenetic protein (BMP) signaling has been shown to modulate the development of renal fibrosis in animal models of kidney injury, but the downstream mediators are incompletely understood. In wild-type mice, canonical BMP signaling mediated by SMAD1/5/8 transcription factors was constitutively active in healthy renal tubules, transiently down-regulated after ischemia reperfusion injury (IRI), and reactivated during successful tubular regeneration. We then induced IRI in mice with a tubular-specific BMP receptor 1A (BMPR1A) deletion. These mice failed to reactivate SMAD1/5/8 signaling in the post-ischemic phase and developed renal fibrosis after injury. Using unbiased genomic analyses, we identified three genes encoding inhibitor of DNA-binding (ID) proteins (Id1, Id2, and Id4) as key targets of BMPR1A-SMAD1/5/8 signaling. BMPR1A-deficient mice failed to re-induce these targets following IRI. Instead, BMPR1A-deficiency resulted in activation of pro-fibrotic signaling proteins that are normally repressed by ID proteins, namely, p38 mitogen-activated protein kinase and cell cycle inhibitor p27. These data indicate that the post-ischemic activation of canonical BMP signaling acts endogenously to repress pro-fibrotic signaling in tubular cells and may help to prevent the progression of acute kidney injury to chronic kidney disease. Acute kidney injury (AKI) constitutes a strong risk factor for progressive chronic kidney disease (CKD).1Chawla L.S. Eggers P.W. Star R.A. et al.Acute kidney injury and chronic kidney disease as interconnected syndromes.N Engl J Med. 2014; 371: 58-66Crossref PubMed Scopus (1140) Google Scholar Although the kidney has a remarkable potential for repair, repeated and severe bouts of AKI are associated with maladaptive repair and progression to tubulointerstitial fibrosis, a universal hallmark of CKD.1Chawla L.S. Eggers P.W. Star R.A. et al.Acute kidney injury and chronic kidney disease as interconnected syndromes.N Engl J Med. 2014; 371: 58-66Crossref PubMed Scopus (1140) Google Scholar, 2Ferenbach D.A. Bonventre J.V. Mechanisms of maladaptive repair after AKI leading to accelerated kidney ageing and CKD.Nat Rev Nephrol. 2015; 11: 264-276Crossref PubMed Scopus (445) Google Scholar, 3Grgic I. Campanholle G. Bijol V. et al.Targeted proximal tubule injury triggers interstitial fibrosis and glomerulosclerosis.Kidney Int. 2012; 82: 172-183Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, 4Bonventre J. Yang L. Cellular pathophysiology of ischemic acute kidney injury.J Clin Invest. 2011; 121: 4210-4221Crossref PubMed Scopus (1326) Google Scholar The molecular pathways that mediate restitutio ad integrum (vs. irreversible structural injury) are under intensive investigation because they constitute potential targets for pharmacological intervention.1Chawla L.S. Eggers P.W. Star R.A. et al.Acute kidney injury and chronic kidney disease as interconnected syndromes.N Engl J Med. 2014; 371: 58-66Crossref PubMed Scopus (1140) Google Scholar, 2Ferenbach D.A. Bonventre J.V. Mechanisms of maladaptive repair after AKI leading to accelerated kidney ageing and CKD.Nat Rev Nephrol. 2015; 11: 264-276Crossref PubMed Scopus (445) Google Scholar In this regard, the BMP signaling pathway has been extensively studied.5Meng X.M. Chung A.C.K. Lan H.Y. Role of the TGF-β/BMP-7/Smad pathways in renal diseases.Clin Sci. 2013; 124: 243-254Crossref PubMed Scopus (282) Google Scholar BMPs are members of the transforming growth factor-β superfamily and signal via binding to type II BMP receptors. Type II BMP receptors then oligomerize and transphosphorylate type I BMP receptors BMPR1A and BMP receptor 1B, which in turn recruit and phosphorylate receptor-regulated SMAD proteins SMAD1, SMAD5, and SMAD8. Phosphorylated SMAD1/5/8 (pSMAD1/5/8) proteins form oligomeric complexes with SMAD4, the mammalian co-Smad, to enter the nucleus and regulate specific target gene transcription.6Wakefield L.M. Hill C.S. Beyond TGFβ: roles of other TGFβ superfamily members in cancer.Nat Rev Cancer. 2013; 13: 328-341Crossref PubMed Scopus (287) Google Scholar, 7Feng X.-H. Derynck R. Specificity and versatility in TGF-β signaling through Smads.Annu Rev Cell Dev Biol. 2005; 21: 659-693Crossref PubMed Scopus (1528) Google Scholar, 8Shi Y. Massagué J. Mechanisms of TGF-β signaling from cell membrane to the nucleus.Cell. 2003; 113: 685-700Abstract Full Text Full Text PDF PubMed Scopus (4800) Google Scholar, 9Miyazono K. Maeda S. Imamura T. BMP receptor signaling: transcriptional targets, regulation of signals, and signaling cross-talk.Cytokine Growth Factor Rev. 2005; 16: 251-263Crossref PubMed Scopus (711) Google Scholar Previous studies have indicated an involvement of BMP signaling in renal fibrosis and the progression of CKD. Bone morphogenetic protein 7 (BMP7), and possibly also BMP4, appears to be the major BMP ligand in the kidney.9Miyazono K. Maeda S. Imamura T. BMP receptor signaling: transcriptional targets, regulation of signals, and signaling cross-talk.Cytokine Growth Factor Rev. 2005; 16: 251-263Crossref PubMed Scopus (711) Google Scholar, 10Sugimoto H. LeBleu V.S. Bosukonda D. et al.Activin-like kinase 3 is important for kidney regeneration and reversal of fibrosis.Nat Med. 2012; 18: 396-404Crossref PubMed Scopus (182) Google Scholar, 11Miyazaki Y. Oshima K. Fogo A. et al.Evidence that bone morphogenetic protein 4 has multiple biological functions during kidney and urinary tract development.Kidney Int. 2003; 63: 835-844Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 12Oxburgh L. Dudley A.T. Godin R.E. et al.BMP4 substitutes for loss of BMP7 during kidney development.Dev Biol. 2005; 286: 637-646Crossref PubMed Scopus (60) Google Scholar BMP7 when applied exogenously has protective effects in the injured kidney progressing to fibrosis.13Zeisberg M. Hanai J. Sugimoto H. et al.BMP-7 counteracts TGF-beta1-induced epithelial-to-mesenchymal transition and reverses chronic renal injury.Nat Med. 2003; 9: 964-968Crossref PubMed Scopus (1181) Google Scholar, 14Zeisberg M. Shah A.A. Kalluri R. Bone morphogenic protein-7 induces mesenchymal to epithelial transition in adult renal fibroblasts and facilitates regeneration of injured kidney.J Biol Chem. 2005; 280: 8094-8100Crossref PubMed Scopus (262) Google Scholar, 15Li R.X. Yiu W.H. Tang S.C.W. Role of bone morphogenetic protein-7 in renal fibrosis.Front Physiol. 2015; 6: 1-9Crossref PubMed Scopus (54) Google Scholar BMP7 reduces the inflammatory infiltrate in models of ischemic AKI and obstructive nephropathy5Meng X.M. Chung A.C.K. Lan H.Y. Role of the TGF-β/BMP-7/Smad pathways in renal diseases.Clin Sci. 2013; 124: 243-254Crossref PubMed Scopus (282) Google Scholar, 16Vukicevic S. Basic V. Rogic D. et al.Osteogenic protein-1 (bone morphogenetic protein-7) reduces severity of injury after ischemic acute renal failure in rat.J Clin Invest. 1998; 102: 202-214Crossref PubMed Scopus (280) Google Scholar, 17Hruska K.A. Guo G. Wozniak M. et al.Osteogenic protein-1 prevents renal fibrogenesis associated with ureteral obstruction.Am J Physiol Renal Physiol. 2000; 279: F130-F143Crossref PubMed Google Scholar and prevents the development of fibrosis.5Meng X.M. Chung A.C.K. Lan H.Y. Role of the TGF-β/BMP-7/Smad pathways in renal diseases.Clin Sci. 2013; 124: 243-254Crossref PubMed Scopus (282) Google Scholar, 18Kalluri R. Zeisberg M. Exploring the connection between chronic renal fibrosis and bone morphogenic protein-7.Histol Histopathol. 2003; 18: 217-224PubMed Google Scholar Endogenous BMP7 activity is downregulated in the kidney after ischemic injury.10Sugimoto H. LeBleu V.S. Bosukonda D. et al.Activin-like kinase 3 is important for kidney regeneration and reversal of fibrosis.Nat Med. 2012; 18: 396-404Crossref PubMed Scopus (182) Google Scholar, 16Vukicevic S. Basic V. Rogic D. et al.Osteogenic protein-1 (bone morphogenetic protein-7) reduces severity of injury after ischemic acute renal failure in rat.J Clin Invest. 1998; 102: 202-214Crossref PubMed Scopus (280) Google Scholar, 19Simon M. Maresh J.G. Harris S.E. et al.Expression of bone morphogenetic protein-7 mRNA in normal and ischemic adult rat kidney.Am J Physiol. 1999; 276: F382-F389PubMed Google Scholar, 20Larman B.W. Karolak M.J. Adams D.C. et al.Chordin-like 1 and twisted gastrulation 1 regulate BMP signaling following kidney injury.J Am Soc Nephrol. 2009; 20: 1020-1031Crossref PubMed Scopus (30) Google Scholar A critical role of the BMP receptor BMPR1A in the injured kidney was previously suggested in mice with γ-glutamyl transpeptidase-Cre–induced constitutive deletion of BMPR1A in proximal tubules.10Sugimoto H. LeBleu V.S. Bosukonda D. et al.Activin-like kinase 3 is important for kidney regeneration and reversal of fibrosis.Nat Med. 2012; 18: 396-404Crossref PubMed Scopus (182) Google Scholar These mice showed an increased susceptibility to the development of renal fibrosis in a nephrotoxic nephritis model.10Sugimoto H. LeBleu V.S. Bosukonda D. et al.Activin-like kinase 3 is important for kidney regeneration and reversal of fibrosis.Nat Med. 2012; 18: 396-404Crossref PubMed Scopus (182) Google Scholar However, whether this finding extends to other models of kidney injury and how loss of BMPR1A signaling leads to increased fibrosis are not fully understood.21Weiskirchen R. Meurer S.K. Gressner O.A. et al.BMP-7 as antagonist of organ fibrosis.Front Biosci. 2009; 14: 4992-5012Crossref PubMed Scopus (66) Google Scholar Here, we present evidence that postischemic activation of BMPR1A-SMAD1/5/8 signaling in renal tubular epithelia acts endogenously to repress profibrotic pathways and restricts the progression of AKI to CKD. Our findings could have therapeutic implications. We used a unilateral IRI mouse model that allowed us to assess the time course of renal regeneration. We observed marked renal tissue injury at 6, 24 and 48 hours after IRI, which significantly improved on day 7, indicating the induction of successful renal recovery (Figure 1a and b; Supplementary Figure S1A). In parallel, we examined nuclear expression of pSMAD1/5/8, which are the downstream effectors of canonical BMP signaling. We found widespread nuclear pSMAD1/5/8 expression in tubular epithelial cells of untreated mouse kidneys (Figure 1c). The majority of pSMAD1/5/8-positive cells were within proximal tubules, which was corroborated by immunostaining for the proximal tubule marker aquaporin 1 (AQP1) (Figure 1c). However, nuclear pSMAD1/5/8 in these cells strongly decreased between 6 and 48 hours after IRI (Figure 1c and d), but partially recovered 7 days after IRI. pSMAD1/5/8 modulation was confirmed by quantification of pSMAD1/5/8 expression in whole kidney extracts after IRI (Figure 1e and f), which also showed an inverse expression time course compared to kidney injury biomarkers lipocalin 2 (LCN2) (neutrophil gelatinase-associated lipocalin [NGAL]) and hepatitis A virus cellular receptor 1 (HAVCR1) (kidney injury molecule 1 [KIM1]) (Figure 1g–i; Supplementary Figure S1B).23Paragas N. Qiu A. Zhang Q. et al.The Ngal reporter mouse detects the response of the kidney to injury in real time.Nat Med. 2011; 17: 216-222Crossref PubMed Scopus (329) Google Scholar, 24Humphreys B.D. Xu F. Sabbisetti V. et al.Chronic epithelial kidney injury molecule-1 expression causes murine kidney fibrosis.J Clin Invest. 2013; 123: 4023-4035Crossref PubMed Scopus (231) Google Scholar These data indicate that IRI-induced AKI is accompanied by a transient downregulation of canonical BMP signaling in tubular epithelia during the acute injury phase (6–48 hours post-IRI), followed by reinduction of BMP-SMAD1/5/8 signaling during recovery (7 days post-IRI). To highlight the role of BMP signaling in the postischemic kidney, we generated a conditional BMP receptor 1a (Bmpr1a) knockout mouse model. In the mature mouse kidney, BMPR1A expression was most pronounced in the outer stripe of the outer medulla (Figure 2a), where it was coexpressed with AQP1 in the proximal tubules (S3 segment), but not with Na-K-Cl cotransporter (NKCC2), a marker of the thick ascending limb of the loop of Henle (Figure 2a). Lower levels of BMPR1A staining were observed in other tubular segments, including cortical proximal tubules. Because global homozygous deletion of Bmpr1a is embryonic lethal and constitutive kidney tubular-specific deletion of Bmpr1a may affect renal development, homeostasis, or renal tubular injury susceptibility,10Sugimoto H. LeBleu V.S. Bosukonda D. et al.Activin-like kinase 3 is important for kidney regeneration and reversal of fibrosis.Nat Med. 2012; 18: 396-404Crossref PubMed Scopus (182) Google Scholar, 25Mishina Y. Suzuki A. Ueno N. et al.Bmpr encodes a type I bone morphogenetic protein receptor that is essential for gastrulation during mouse embryogenesis.Genes Dev. 1995; 9: 3027-3037Crossref PubMed Scopus (641) Google Scholar we conditionally targeted Bmpr1a expression in adult renal tubules. For this purpose, we used a doxycycline (dox)-inducible tubular-specific Cre line (Pax8rtTA;LC-1).26Traykova-Brauch M. Schönig K. Greiner O. et al.An efficient and versatile system for acute and chronic modulation of renal tubular function in transgenic mice.Nat Med. 2008; 14: 979-984Crossref PubMed Scopus (198) Google Scholar To test Cre recombination efficiency in renal tubules, we first analyzed β-galactosidase activity in dox-treated Pax8rtTA;LC-1;ROSA26R reporter mice, which revealed strong X-Gal staining in renal tubules (Supplementary Figure S2A and B). Dox-inducible Bmpr1a conditional knockout mice were then generated by mating Bmpr1afl/fl with Pax8rtTA;LC-1 mice to obtain Pax8rtTA;LC-1;Bmpr1afl/fl offspring (Figure 2b).27Mishina Y. Hanks M.C. Miura S. et al.Generation of Bmpr/Alk3 conditional knockout mice.Genesis. 2002; 32: 69-72Crossref PubMed Scopus (220) Google Scholar Treatment with dox was performed for 14 days in 3-week-old mice, followed by 3 to 4 weeks without dox. This procedure induced Bmpr1a exon 2 deletion as demonstrated by polymerase chain reaction (PCR) using renal genomic DNA and cDNA and by RNA sequencing (RNA-seq) (Figure 2c and d). However, in healthy adult kidneys, BMPR1A protein was still detected in the tubules of dox-treated Pax8rTA;LC-1;Bmpr1afl/fl (Bmpr1a cKO) mice, which was presumably due to the low proliferation rate of renal tubular cells and to a long half-life of BMPR1A protein (sham-untreated control [Ctrl] and Bmpr1a cKO) (Figure 2e–h).28Thomasova D. Anders H.J. Cell cycle control in the kidney.Nephrol Dial Transplant. 2015; 30: 1622-1630Crossref PubMed Scopus (64) Google Scholar IRI is known to induce massive turnover in renal tubular cells,29Humphreys B.D. Valerius M.T. Kobayashi A. et al.Intrinsic epithelial cells repair the kidney after injury.Cell Stem Cell. 2008; 2: 284-291Abstract Full Text Full Text PDF PubMed Scopus (678) Google Scholar which is why we hypothesized that Bmpr1a cKO mice would fail to generate BMPR1A-positive proximal tubules in the postischemic phase whereas the regenerated proximal tubules of dox-treated Ctrl littermates would express de novo BMPR1A. We therefore analyzed BMPR1A expression in Bmpr1a cKO and Ctrl mice 7 days after IRI. At this time point, BMP signaling is normally reinduced in tubular cells of wild-type kidneys (Figure 1c and e–g). Consistent with our hypothesis, tubular BMPR1A expression on day 7 after IRI was low or absent in Bmpr1a cKO kidneys but was reinduced in Ctrl kidneys (Figure 2e and f). Reduction of BMPR1A protein in Bmpr1a cKO kidneys was confirmed by Western blotting (Figure 2g and h). Thus, our model induced specific deletion of BMPR1A protein in tubular cells of the kidney during recovery from IRI. Therefore, this model allowed us to selectively assess the effect of post-IRI BMPR1A activation when comparing Bmpr1a cKO mice with Ctrl mice. To analyze the effect of tubular BMPR1A on SMAD1/5/8 phosphorylation, we analyzed BMPR1A-dependent pSMAD1/5/8 activation after IRI. As expected, nuclear pSMAD1/5/8 expression was reactivated 7 days after injury in renal tubules of ischemic Ctrl kidneys but remained low in Bmpr1a cKO tubules (Figure 2i–l). In contrast, nuclear pSMAD1/5/8 remained detectable in CD31-positive endothelial cells in both Ctrl and Bmpr1a cKO kidneys 7 days after injury (Figure 2m and n). These observations indicated that reactivation of tubular pSMAD1/5/8 in regenerating kidneys was specifically dependent on BMPR1A in tubular epithelial cells. Because BMP signaling is downregulated in the initial phase after IRI, we did not expect any effect of BMPR1A inactivation on AKI. This expectation was confirmed by measuring urinary excretion of NGAL 24 hours after IRI, which showed no difference between Bmpr1a cKO and Ctrl mice (Supplementary Figure S3A). In addition, no difference in renal Ngal and Kim1 mRNA expression was observed when comparing Bmpr1a cKO mice with Ctrl mice 48 hours after IRI (Supplementary Figure S3B and C). Histological grading at 48 hours after IRI revealed evidence of tubular injury in Bmpr1a cKO mice similar to Ctrl mice (Supplementary Figure S3D and E). In fact, at early postischemic time points none of the morphological parameters we evaluated displayed differences between Bmpr1a cKO and Ctrl mice (Supplementary Figure S3F). Although tubular proliferation is most pronounced 48 hours after IRI,30Berger K. Moeller M.J. Mechanisms of epithelial repair and regeneration after acute kidney injury.Semin Nephrol. 2014; 34: 394-403Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 31Berger K. Bangen J.M. Hammerich L. et al.Origin of regenerating tubular cells after acute kidney injury.Proc Natl Acad Sci U S A. 2014; 111: 1533-1538Crossref PubMed Scopus (112) Google Scholar tubular KI67 expression revealed no significant differences between Bmpr1a cKO and Ctrl mice 48 hours after IRI (Supplementary Figure S3G and H). Similarly, DNA fragmentation of renal tubular cells analyzed by terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling assay, indicative of various types of cell death, displayed no differences between Bmpr1a cKO and Ctrl mice (Supplementary Figure S3I and J). Together, these data confirm that loss of tubular BMPR1A did not significantly affect the early postischemic phase after IRI. We next examined renal histology of Bmpr1a cKO and Ctrl mice on day 7 after IRI, the time point when BMPR1A-SMAD1/5/8 signaling is observed to be reactivated in Ctrl mice and renal recovery is taking place (Figure 1). Seven days after IRI, Bmpr1a cKO kidneys revealed increased tubular damage as compared with ischemic Ctrl kidneys, as quantified by total tubular injury score (Figure 3a and b). Most of the evaluated morphological parameters showed a significant worsening in Bmpr1a cKO kidneys compared with Ctrl kidneys (Figure 3c). As tubular injury is predominantly triggered by inflammatory mechanisms at this time point,32Jang H.R. Rabb H. Immune cells in experimental acute kidney injury.Nat Rev Nephrol. 2014; 11: 88-101Crossref PubMed Scopus (281) Google Scholar, 33Huen S.C. Cantley L.G. Macrophages in renal injury and repair.Annu Rev Physiol. 2017; 79: 449-469Crossref PubMed Scopus (170) Google Scholar we assessed the interstitial infiltration of macrophages (F4/80) and neutrophils (Ly6B.2) by immunofluorescence staining. In Bmpr1a cKO kidneys, the number of infiltrating macrophages and neutrophils was increased by 51% and 59%, respectively, on day 7 after IRI as compared with Ctrl mice (Figure 3d–g). However, we observed no differences in proliferation or cell death 7 days after IRI between Bmpr1a cKO and Ctrl kidneys (Supplementary Figure S4). We next assayed evidence of permanent tissue remodeling and tubulointerstitial fibrosis in these mice. Capillary rarefaction, a hallmark of CKD,34Bonventre J V. Primary proximal tubule injury leads to epithelial cell cycle arrest, fibrosis, vascular rarefaction, and glomerulosclerosis.Kidney Int Suppl (2011). 2014; 4: 39-44Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar already became evident on day 7 after IRI by a reduction of CD31-positive endothelial cells in Bmpr1a cKO kidneys compared with Ctrl kidneys (Figure 3h and i). Twenty-one days after IRI, Bmpr1a cKO kidneys displayed markedly aggravated renal fibrosis as compared with Ctrl mice, as shown by increased Masson trichrome staining and collagen IV protein expression (Figure 3j–m). Overall, these data indicated that after IRI, inactivation of endogenous BMPR1A-SMAD1/5/8 signaling in renal tubules exacerbated inflammation and tubular injury during the recovery phase and promoted progressive tubulointerstitial fibrosis in the long term. To identify target genes of BMP-SMAD1/5/8 signaling, we developed a pSMAD1/5/8 chromatin immunoprecipitation (ChIP) protocol using Ctrl kidneys harvested 7 days after IRI when pSMAD1/5/8 is reactivated. To obtain a global pSMAD1/5/8 DNA-binding profile, we used next-generation sequencing (chromatin immunoprecipitation and sequencing [ChIP-seq]). After peak calling using the model-based analysis of ChIP-seq tool, we identified 839 pSMAD1/5/8-specific peaks (Supplementary Table S1). Peak annotation revealed that peaks were overrepresented in the vicinity of genes and their transcriptional start sites (Figure 4a). The Gene Ontology analysis of the peak-associated genes revealed an overrepresentation of transforming growth factor-β and BMP signaling pathway members (Figure 4b). The findings included all 4 ID family members (Id1, Id2, Id3, and Id4) and the inhibitory Smads Smad6 and Smad7 (Figure 4c; Supplementary Figure S5A). These data were confirmed by ChIP-qPCR assays (Figure 4d; Supplementary Figure S5B). We next obtained global transcriptional profiles from Bmpr1a cKO and Ctrl kidneys 7 days after IRI by using RNA-seq. This analysis revealed 61 differentially expressed genes (absolute fold change, >1.4; P < 0.01), of which 31 were upregulated and 30 downregulated in Bmpr1a cKO kidneys compared with Ctrl kidneys (Figure 4e; Supplementary Table S2). The Gene Ontology analysis of downregulated genes revealed an overrepresentation of transforming growth factor-β/BMP and ID signaling pathways (Figure 4f), and quantitative real-time PCR (qPCR) confirmed Id1, Id2, and Id4 mRNA downregulation in Bmpr1a cKO kidneys compared with Ctrl kidneys 7 days after IRI (Figure 4h; Supplementary Figure S5C). The Gene Ontology analysis of genes upregulated in Bmpr1a cKO kidneys highlighted classes related to cytokine and growth factor activity and connective tissue development (Figure 4g), supporting the Bmpr1a cKO phenotype observed on 7 days after IRI (Figure 3). Real-time qPCR analysis confirmed the mRNA overexpression of Ccl7, Ccl20, Csf, and Lif cytokines and of platelet-derived growth factor B (Pdgfb) profibrotic growth factor mRNAs in ischemic Bmpr1a cKO kidneys compared with Ctrl kidneys (Figure 4i). We then compared pSMAD1/5/8 ChIP-seq data from Ctrl kidneys with differential expression data from Bmpr1a cKO vs. Ctrl kidneys 7 days after IRI to identify putative direct targets that are activated or repressed by pSMAD1/5/8. Surprisingly, this comparison yielded a small number of direct pSMAD1/5/8 target genes. Among the genes with the promoter-proximal association of pSMAD1/5/8 (±2.5 kb from the gene transcription start site), only the ID family members Id1, Id2, and Id4 were differentially expressed in ischemic Bmpr1a cKO versus Ctrl kidneys (Figure 4c, d, and h). The most pronounced downregulation was observed for Id4 (Figure 4h). In addition, the time course of Id4 expression after IRI (and to a lesser degree also Id1 and Id2) in wild-type kidneys closely mirrored pSMAD1/5/8 activity, which was downregulated at early time points after IRI, but markedly upregulated 7 days after IRI (Figure 4j; see also Figure 1d and e). Western blot analysis indicated a decrease of ID4 protein in Bmpr1a cKO kidneys compared with Ctrl kidneys 7 days after IRI (Figure 4k and l). Together, these results indicate that Id1, Id2, and Id4 genes are direct targets of BMP-BMPR1A-SMAD1/5/8 signaling, with Id4 displaying the strongest pSMAD1/5/8-dependent regulation. Previous studies identified P38 mitogen-activated kinase (P38 MAPK) as a key target that was repressed by ID4 or ID2 in epithelial cells of the mammary gland or pancreas, respectively.35Dong J. Huang S. Caikovski M. et al.ID4 regulates mammary gland development by suppressing p38MAPK activity.Development. 2011; 138: 5247-5256Crossref PubMed Scopus (34) Google Scholar, 36Hua H. Sarvetnick N. ID2 promotes the expansion and survival of growth-arrested pancreatic beta cells.Endocrine. 2007; 32: 329-337Crossref PubMed Scopus (7) Google Scholar Moreover, P38 MAPK had previously been associated with the development of renal fibrosis. P38 MAPK is expressed in the renal tubular epithelium, and treatment with a P38 MAPK inhibitor protected rats from fibrosis after unilateral ureteral obstruction.37Stambe C. The role of p38 mitogen-activated protein kinase activation in renal fibrosis.J Am Soc Nephrol. 2004; 15: 370-379Crossref PubMed Scopus (171) Google Scholar We therefore hypothesized that BMP-SMAD1/5/8 activation of ID proteins would repress P38 MAPK activation. In line with this hypothesis, Western blot analyses indicated that phosphorylated P38 (p-P38) MAPK expression was strongly upregulated in kidneys of Bmpr1a cKO mice compared with Ctrl mice 7 days after IRI, which coincided with the failure to reactivate ID4 (Figure 5a–c). Immunofluorescence staining for p-P38 indicated activation of the P38 MAPK pathway in AQP1-positive proximal tubular epithelial cells in Bmpr1a cKO mice 7 days after IRI whereas no such activation was observed in Ctrl mice at the same time point (Figure 5d and e). In other model systems, P38 MAPK stabilizes the cell cycle regulator cyclin-dependent kinase inhibitor 1B P27.35Dong J. Huang S. Caikovski M. et al.ID4 regulates mammary gland development by suppressing p38MAPK activity.Development. 2011; 138: 5247-5256Crossref PubMed Scopus (34) Google Scholar, 38Kim J. Wong P.K.Y. Loss of ATM impairs proliferation of neural stem cells through oxidative stress-mediated p38 MAPK signaling.Stem Cells. 2009; 27: 1987-1998Crossref PubMed Scopus (105) Google Scholar, 39Cuadrado M. Gutierrez-Martinez P. Swat A. et al.P27Kip1 stabilization is essential for the maintenance of cell cycle arrest in response to DNA damage.Cancer Res. 2009; 69: 8726-8732Crossref PubMed Scopus (50) Google Scholar P27 is upregulated in kidney fibrosis and is a pivotal participant in the pathogenesis of progressive fibrosis.40Lovisa S. LeBleu V.S. Tampe B. et al.Epithelial-to-mesenchymal transition induces cell cycle arrest and parenchymal damage in renal fibrosis.Nat Med. 2015; 21: 998-1009Crossref PubMed Scopus (583) Google Scholar, 41Wolf G. Schanze A. Stahl R.A.K. et al.p27Kip1 knockout mice are protected from diabetic nephropathy: evidence for p27Kip1 haplotype insufficiency.Kidney Int. 2005; 68: 1583-1589Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 42Chkhotua A.B. Abendroth D. Froeba G. et al.Up-regulation of cell cycle regulatory genes after renal ischemia/reperfusion: differential expression of p16(INK4a), p21 (WAF1/CIP1) and p27(Kip1) cyclin-dependent kinase inhibitor genes depending on reperfusion time.Transpl Int. 2006; 19: 72-77Crossref PubMed Scopus (35) Google Scholar Consistently, we found that P27 was upregulated 7 days after IRI in Bmpr1a cKO kidneys compared with Ctrl kidneys (Figure 5f and g). Immunofluorescence staining indicated nuclear P27 expression in AQP1-positive proximal tubular cells 7 days after IRI in Bmpr1a cKO kidneys, but not in Ctrl kidneys (Figure 5h and i). Together, these data indicated that the inhibition of BMPR1A-SMAD1/5/8-ID signaling in tubular epithelia after AKI in Bmpr1a cKO mice was associated with ectopic activation of p-P38 MAPK and P27. Our study demonstrates an essential role for BMPR1A-SMAD1/5/8 signaling in renal tubular regeneration after IRI. We identified the genes encoding ID1, ID2, and ID4 as central direct targets of tubular epithelial BMP-SMAD1/5/8 signaling and observed activation of P38 MAPK and P27 in BMPR1A-deficient kidneys during the early repair phase 7 days after IRI. These results suggest a novel molecular cascade downstream of BMP signaling in renal epithelia in vivo (Figure 6). Inhibition of postischemic BMPR1A signaling was also associated with increased tubular injury scores during the early repair phase, together with incre" @default.
• W2901029372 created "2018-11-29" @default.
• W2901029372 creator A5030750239 @default.
• W2901029372 creator A5056429688 @default.
• W2901029372 creator A5062793291 @default.
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• W2901029372 date "2019-01-01" @default.
• W2901029372 modified "2023-10-02" @default.
• W2901029372 title "Canonical BMP signaling in tubular cells mediates recovery after acute kidney injury" @default.
• W2901029372 cites W1582760249 @default.
• W2901029372 cites W1768274470 @default.
• W2901029372 cites W1965699247 @default.
• W2901029372 cites W1967447462 @default.
• W2901029372 cites W1970377223 @default.
• W2901029372 cites W1971075697 @default.
• W2901029372 cites W1971230349 @default.
• W2901029372 cites W1971799714 @default.
• W2901029372 cites W1980224469 @default.
• W2901029372 cites W1986240011 @default.
• W2901029372 cites W1989061028 @default.
• W2901029372 cites W1993345123 @default.
• W2901029372 cites W1995338323 @default.
• W2901029372 cites W1999265533 @default.
• W2901029372 cites W2003599465 @default.
• W2901029372 cites W2018091219 @default.
• W2901029372 cites W2021341670 @default.
• W2901029372 cites W2031920531 @default.
• W2901029372 cites W2032700434 @default.
• W2901029372 cites W2036755333 @default.
• W2901029372 cites W2041736631 @default.
• W2901029372 cites W2046051277 @default.
• W2901029372 cites W2048899307 @default.
• W2901029372 cites W2050475853 @default.
• W2901029372 cites W2051418621 @default.
• W2901029372 cites W2057267524 @default.
• W2901029372 cites W2057389695 @default.
• W2901029372 cites W2067008993 @default.
• W2901029372 cites W2067366368 @default.
• W2901029372 cites W2068050227 @default.
• W2901029372 cites W2076124674 @default.
• W2901029372 cites W2077967941 @default.
• W2901029372 cites W2079363030 @default.
• W2901029372 cites W2080627387 @default.
• W2901029372 cites W2083893575 @default.
• W2901029372 cites W2087103113 @default.
• W2901029372 cites W2088089995 @default.
• W2901029372 cites W2089953630 @default.
• W2901029372 cites W2105199836 @default.
• W2901029372 cites W2117200840 @default.
• W2901029372 cites W2120006551 @default.
• W2901029372 cites W2128474411 @default.
• W2901029372 cites W2128603782 @default.
• W2901029372 cites W2131271579 @default.
• W2901029372 cites W2134931198 @default.
• W2901029372 cites W2134947948 @default.
• W2901029372 cites W2138207763 @default.
• W2901029372 cites W2140729960 @default.
• W2901029372 cites W2154568166 @default.
• W2901029372 cites W2165695219 @default.
• W2901029372 cites W2166487539 @default.
• W2901029372 cites W2168456732 @default.
• W2901029372 cites W2170551349 @default.
• W2901029372 cites W2171808845 @default.
• W2901029372 cites W2189664213 @default.
• W2901029372 cites W2254511588 @default.
• W2901029372 cites W2343540188 @default.
• W2901029372 cites W2509717357 @default.
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