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Curtailing Endothelial TGF-b Signaling Is Sufficient to Reduce Endothelial-Mesenchymal Transition and Fibrosis in CKD Sandhya Xavier,*† Radovan Vasko,*†‡ Kei Matsumoto,*† Joseph A. Zullo,*† Robert Chen,*† Julien Maizel,*† Praveen N. Chander,§ and Michael S. Goligorsky*† Departments of *Medicine, Pharmacology, Physiology, and §Pathology, and †Renal Research Institute, New York Medical College, Valhalla, New York; and ‡Department of Nephrology and Rheumatology, University Medical Center, Goettingen, Germany
ABSTRACT Excessive TGF-b signaling in epithelial cells, pericytes, or fibroblasts has been implicated in CKD. This list has recently been joined by endothelial cells (ECs) undergoing mesenchymal transition. Although several studies focused on the effects of ablating epithelial or fibroblast TGF-b signaling on development of fibrosis, there is a lack of information on ablating TGF-b signaling in the endothelium because this ablation causes embryonic lethality. We generated endothelium-specific heterozygous TGF-b receptor knockout (TbRIIendo+/2) mice to explore whether curtailed TGF-b signaling significantly modifies nephrosclerosis. These mice developed normally, but showed enhanced angiogenic potential compared with TbRIIendo+/+ mice under basal conditions. After induction of folic acid nephropathy or unilateral ureteral obstruction, TbRIIendo+/2 mice exhibited less tubulointerstitial fibrosis, enhanced preservation of renal microvasculature, improvement in renal blood flow, and less tissue hypoxia than TbRIIendo+/+ counterparts. In addition, partial deletion of TbRII in the endothelium reduced endothelial-to-mesenchymal transition (EndoMT). TGF-b–induced canonical Smad2 signaling was reduced in TbRII+/2 ECs; however, activin receptor-like kinase 1 (ALK1)–mediated Smad1/5 phosphorylation in TbRII+/2 ECs remained unaffected. Furthermore, the S-endoglin/L-endoglin mRNA expression ratio was significantly lower in TbRII+/2 ECs compared with TbRII+/+ ECs. These observations support the hypothesis that EndoMT contributes to renal fibrosis and curtailing endothelial TGF-b signals favors Smad1/5 proangiogenic programs and dictates increased angiogenic responses. Our data implicate endothelial TGF-b signaling and EndoMT in regulating angiogenic and fibrotic responses to injury. J Am Soc Nephrol 26: ccc–ccc, 2014. doi: 10.1681/ASN.2013101137
CKD leading to end stage renal failure is associated with tissue scarring or fibrosis.1 For development of effective therapeutic strategies, it is essential to define the cellular and molecular mechanisms that modulate regeneration of kidney.2 There is strong evidence that TGF-b1 is a key mediator in the pathogenesis of renal fibrosis in both mouse models and human kidney diseases.1–3 Direct evidence for its role in renal fibrosis comes from studies demonstrating that mice overexpressing an active form of TGF-b1 in the liver develop both progressive liver and renal fibrosis.4 Although restoration of adequate microvascular supply is essential for renal regeneration J Am Soc Nephrol 26: ccc–ccc, 2014
after chronic injury, genetic studies have revealed the pivotal role of TGF-b signaling in angiogenesis, as well as vascular integrity.5 Incidentally, loss of
Received October 30, 2013. Accepted June 25, 2014. Published online ahead of print. Publication date available at www.jasn.org. Correspondence: Dr. Sandhya Xavier or Michael S. Goligorsky, Department of Medicine, Renal Research Institute, New York Medical College, 15 Dana Road, Valhalla, NY 10595. Email: [email protected] or [email protected] Copyright © 2014 by the American Society of Nephrology
ISSN : 1046-6673/2604-ccc
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peritubular capillaries, a hallmark of progressive renal disease in humans, plays a role in the etiology of interstitial fibrosis and tubular atrophy.6 TGF-b signal is initiated when the ligand binds to its cognate TGF-b type II receptor (TbRII) leading to phosphorylation of TbRI (also known as ALK5). The activated TbRI kinase phosphorylates canonical Smad2/3, which translocates to the nucleus and controls gene expression to inhibit endothelial proliferation, migration, and capillary tube formation. 7 TGF-b can also bind to activin receptor-like kinase ALK1 exclusively in endothelial cells (ECs), which induces Smad1/5 to potentiate angiogenic programs. Phenotypic and molecular
Figure 1. Characterization of endothelial TbRII heterozygote mice. (A) Genotyping of pups from TGF-bRIIFlox/Flox and Tie2-Cre: TGF-bRIIFlox/WT matings. Tail PCR genotyping analysis. The top panel shows detection of floxed and WT fragments. The bottom panel shows detection of Cre-transgene. (B) Real-time PCR analysis for TbRII mRNA expression in kidney ECs isolated from TbRIIendo+/+ and TbRIIendo+/2 mice (n=4). *P,0.05, TbRII+/+ versus TbRII+/2. (C) Western blot for Smad2 signaling in kidney ECs. WT, wild type; Cont, control.
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characterization of knockout mice for TGF-b signaling components have demonstrated their critical role in angiogenesis and cardiovascular system.8 Mice with ablation of endothelial TbRII or TbRI (ALK5) show vascular defects in the yolk sac and embryonic lethality as early as embryonic day 10.5. The embryos also have severe anemia and defective vasculogenesis,9,10 both signs of impaired differentiation of hemangioblast to hematopoietic stem cells and endothelial progenitor cells.
Figure 2. Functional characterization of TbRII heterozygote mice. (A) Images and quantification of ex vivo angiogenesis assays in three-dimensional matrigel using explant cultures of aortic ring segments at day 6 from TbRIIendo+/+ and TbRIIendo+/2 mice (n=5). (B) Western blot for Smad1/5 signaling in kidney ECs isolated from TbRIIendo+/+ and TbRIIendo+/2 mice. (C) Endoglin S/L mRNA expression ratios in kidney ECs. *P,0.05, TbRII+/+ versus TbRII+/2 and TGF-b1 treated TbRII+/+ versus TbRII+/2. Cont, control.
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Nguyen et al.11 recently showed that deletion of TbRII in ECs results in impaired blood vessel morphogenesis in the brain and intracerebral hemorrhage leading to embryonic lethality. The consequences of the partial ablation of TbRII in ECs remain unexplored. Collectively, these studies confirm that TGF-b signaling in ECs is essential for precise regulation of blood vessel assembly and consequently, normal embryonic development. Although increasing evidence shows the role of TGF-b1 in vasculogenesis or renal inflammation and fibrosis, the role of EC TGF-b receptor signaling in the progression of kidney diseases remains unexplored. The link between endothelial dysfunction and progression of fibrosis awaits elucidation. Several cell types have been proposed as initiators of tubulointerstitial fibrosis, including tubular epithelial cells, pericytes, fibroblasts, and more recently ECs, all of which can undergo mesenchymal transition to form myofibroblasts.12–16
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Indeed, several studies focused on the ablation of epithelial TGF-b signaling or depletion of TbRII in kidney fibroblasts to alter the development of fibrosis.17 The same strategy has been reported for epithelial cells in the proximal tubule and collecting duct.18,19 However, there are no published data on its ablation in the endothelium partly because of the unavailability of endothelial TbRII knockout mice due to the embryonic lethality.9,10 To address the question of whether endothelial TGF-b signals contribute to renal fibrosis, we utilized mice with partial ablation of TbRII in this study (i.e., mice heterozygous for TbRII), and investigated the role of endothelial TGF-b signaling in renal fibrosis of the folic acid nephropathy (FAN) and unilateral ureteral obstruction (UUO) models. The data demonstrate that curtailing TGF-b signaling in vascular ECs is sufficient to reduce endothelial-tomesenchymal transition (EndoMT), improve angiogenesis, and reduce fibrosis.
Figure 3. Fibrosis is reduced in TbRIIendo+/2 mice with FA toxicity. (A) Representative images of Masson’s trichrome–stained kidney sections from 12-week-old TbRIIendo+/+ and TbRIIendo+/2 mice treated with vehicle or FA 6 weeks after injection (n=5). (B) Quantification of fibrotic area (color quantification method). (C) Expression of collagen I and collagen III examined by quantitative real-time PCR. *P,0.05, treated TbRIIendo+/+ versus treated TbRIIendo+/2. Original magnification, 34 and 310. J Am Soc Nephrol 26: ccc–ccc, 2014
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RESULTS Generation of Mice with Endothelial Deletion of TbRII
We intercrossed mice homozygous for the floxed TbRII allele with mice heterozygous for Cre-recombinase under the control of the Tie2-promoter to obtain Tie2-Cre;TbRIIflox/+, which were crossed with TbRIIflox/flox mice. Genotyped offspring from these
intercrosses identified heterozygotes (Tie2-Cre;TbRIIflox/+) and wild-type pups (TbRIIflox/flox or TbRIIflox/+), whereas homozygote pups (Tie2-Cre; TbRIIflox/flox) were embryonic lethal, as previously reported.9–11 Percentages of these genotypes were found to be 32.3% each for TbRIIflox/flox and TbRIIflox/+ mice and 35.5% of Tie2-Cre;TbRIIflox/+ mice. Figure 1A shows results of genotyping PCR analysis of mice. Heterozygous mice
Figure 4. Fibrosis is reduced in TbRIIendo+/2 UUO mice. (A) Representative images of Masson’s trichrome–stained UUO and contralateral kidney sections from TbRIIendo+/+ and TbRIIendo+/2 mice (n=4). (B) Quantification of fibrotic area (color quantification method) *P,0.01, TbRIIendo+/+ UUO versus TbRIIendo+/2 UUO. (C) Representative images for a-SMA staining in UUO and contralateral kidney sections from TbRIIendo+/+ and TbRIIendo+/2 UUO mice. Original magnification, 34 and 310.
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(Tie2-Cre;TbRIIflox/+ hereafter referred to as TbRIIendo+/2) were viable, did not exhibit any phenotypic differences compared with control mice (TbRIIendo+/+), and showed no abnormalities in renal function at 6 weeks of age. We evaluated TbRII expression levels in kidney ECs isolated from TbRIIendo+/+ and TbRIIendo+/2 mice by quantitative real-time PCR analysis. Figure 1B documents the expected significant reduction in mRNA expression of TbRII in isolated ECs from TbRIIendo+/2 mice. When treated with TGF-b1 or TGF-b2 at a concentration of 5 ng/ml for 30 minutes, phosphorylation of Smad2 was reduced in ECs from TbRIIendo+/2 mice (Figure 1C). To ensure endothelial origin of isolated ECs, we stained cells for the expression of endothelial markers and showed that .90% of cells expressed VE-cadherin (CD144) and platelet-EC adhesion molecules (CD31) (Supplemental Figure 1). These data provided essential information on mice with partial endothelial ablation of TbRII signaling, demonstrating their viability, as opposed to total ablation, and normal development under unstressed conditions. Partial Endothelial Ablation of TbRII Signaling Boosts Ex Vivo Angiogenic Potential and Favors L-Endoglin– Mediated Smad1/5 Signaling
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because it is purely tubulopathic and does not affect ECs. Serum creatinine levels and urinary albumin/creatinine ratio were significantly increased in both TbRIIendo+/+ and TbRIIendo+/2 mice 48 hours after folic acid (FA) injection (Supplemental Figure 2, B and C). Both groups of mice showed frequent attenuation and focal loss of brush borders, focal mild- to-moderate tubular dilation, simplification, and rare necrosis of tubular epithelium. Granular eosinophilic material was observed in tubular profiles suggesting necrotic debris and was more prominent in TbRIIendo+/2 group (Supplemental Figure 2A). In contrast with our observations in acute renal injury, during chronic phase of injury, interstitial fibrosis was significantly ameliorated in the TbRIIendo+/2 +FA group compared with the TbRIIendo+/+ +FA group (Figure 3A). Consistent with these findings, expression of mRNA encoding collagen I and III was markedly increased in the TbRIIendo+/+ +FA group, whereas upregulation of these genes was ameliorated in the TbRIIendo+/2 +FA group of mice (Figure 3C). In a UUO model, a similar observation of reduced fibrosis in TbRIIendo+/2 mice was made (Figure 4B). Furthermore, the medullary region of UUO kidneys from TbRIIendo+/+ mice showed striking fibrosis, which
We evaluated the angiogenic potential using an ex vivo aortic endothelial sprouting assay.20 Data demonstrated that thoracic aortic rings obtained from TbRIIendo+/2 mice had accelerated sprouting in three-dimensional matrigel assays, which became clearly detectable by day 6 (Figure 2A). To prove that it was attributable to TGF-b signaling, we next examined the Smad1/5 profile in ECs isolated from kidneys of TbRIIendo+/+ and TbRIIendo+/2 mice, because ALK1-induced Smad1/5 signaling is proangiogenic, as opposed to the Smad2/3 pathway, which inhibits proliferation and migration of ECs. Smad1/5 levels at baseline were slightly reduced in ECs from TbRIIendo+/2 mice; nevertheless, after TGF-b1 or TGF-b2 treatment, phosphorylation of Smad1/5 was not impaired in ECs from TbRIIendo+/2 mice (Figure 2B). The expression ratio of short/long (S/L) isoforms of the auxiliary TGF-b1 receptor endoglin was significantly lower in ECs from TbRIIendo+/2 mice at baseline and after TGF-b1 treatment (Figure 2C). An increase in the S/L ratio of endoglin is known to be associated with senescence of ECs.21 Our observation of a reduced S/L ratio in ECs from TbRIIendo+/2 mice suggests that L-endoglin–mediated Smad1/5 proangiogenic signaling is more pronounced and increases angiogenic response, as seen ex vivo in TbRIIendo+/2 mice. This difference in angiogenic competence between TbRIIendo+/2 and TbRIIendo+/+ mice could play a role in vivo after imposition of stress, as was examined in the next series of experiments. Impaired Endothelial TbRII Signaling Improves Angiogenesis and Ameliorates Fibrotic Response in Chronic Kidney Injury
The FAN model was used to study the effects of acute and chronic injury in mice with partial deletion of TbRII in the endothelium. This model is advantageous for the study, J Am Soc Nephrol 26: ccc–ccc, 2014
Figure 5. Ex vivo angiogenesis assays in mice with FA nephrotoxicity. (A) Representative images of sprouting capillary cords in aortic explants at day 13. (B) Quantitative angiogenesis analysis in TbRIIendo+/+ or TbRIIendo+/2 vehicle or FA-treated animals. *P,0.01 untreated versus treated TbRIIendo+/+ or untreated versus treated TbRIIendo+/2.
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was ameliorated in the TbRIIendo+/2 group (Figure 4A). Consistent with this, a-smooth muscle actin (a-SMA) staining in the medulla was much more enhanced in UUO kidneys from
TbRIIendo+/+ mice compared with TbRIIendo+/2 mice (Figure 4C). Thoracic aortas were obtained from FAN mice to study their ex vivo angiogenic potential. Results showed that vessels obtained from mice heterozygous for endothelial TbRII have better angiogenic potential when challenged with FA compared with TbRIIendo+/+ mice, in which angiogenesis was found to be inhibited during FAinduced renal fibrosis (Figure 5). The impaired angiogenic response in TbRIIendo+/+ mice with FAN was associated with a significant loss of patent and functional capillaries in the kidney as judged by capillary density measurement using intravitally injected Lycopersicon esculentum (lectin) to label functional vessels, compared with CD31-positive total microvasculature in the kidneys (Figure 6A). Although CD31 staining was comparable in all groups of mice, lectin reporting only functional microvessels showed a dramatic decline in TbRIIendo+/+ mice with FAN, but TbRIIendo+/2 mice with FAN had a much higher proportion of functional microvessels. The mismatch between patent and total vessels also suggests initiation of an active EndoMT program after renal injury and that curtailed TGF-b signaling in the endothelium may restrain EndoMT transition, as addressed in the next series of experiments. In order to investigate the mechanisms underlying loss of patency of microvessels and to see whether this causes hypoperfusion and consequently tissue hypoxia, we performed laser-Doppler flowmetry of renal blood flow in the cortical and medullary regions of UUO kidneys. As shown in Figure 7, renal blood flow was impaired in the medulla of TbRIIendo+/+ mice, whereas flow in TbRIIendo+/2 mice was significantly preserved. We also probed kidneys for formation of pimonidazole adducts to measure regions with ,10 mmHg pO2 as represented in Figure 7C. A significant difference in staining was observed between both groups of mice with TbRIIendo+/+ mice displaying higher hypoxic regions compared with Figure 6. Microvascular density (CD31) and patency (lectin) in mice with FA neph- TbRIIendo+/2 mice.
rotoxicity. (A) Representative images for CD31 (green) and lectin (red) staining (n=5). (B) Average lengths (in micrometers) of CD31- or lectin-positive peritubular capillaries per image. (C) Ratios of average lengths of lectin- to CD31-positive capillaries (percentage) per image. *P,0.01, treated TbRIIendo+/+ versus TbRIIendo+/2 lectin-positive capillary length; **P,0.001, control versus FA-treated TbRIIendo+/+ mice; #P,0.01, treated TbRIIendo+/+ versus TbRIIendo+/2; N.S., P=NS for control versus FA-treated TbRIIendo+/2 mice. Original magnification, 340. Per the journal style, P values were rounded to two decimal places.
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Curtailed Endothelial TbRII Signaling Significantly Reduces EndoMT in FAN and UUO Injury
We investigated whether impaired TGF-b receptor signaling in the endothelium affects EndoMT processes during renal fibrosis. To J Am Soc Nephrol 26: ccc–ccc, 2014
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Figure 7. Renal blood flow measurement and pimonidazole staining in kidneys of TbRIIendo+/+ and TbRIIendo+/2 UUO mice. (A) Representative image scans from laserDoppler flowmetry analysis of contralateral and UUO kidneys from TbRIIendo+/+ and TbRIIendo+/2 mice (n=3). (B) Quantification of cortex and medullary renal blood flow in kidneys. *P,0.001, TbRIIendo+/+ and TbRIIendo+/2 UUO mice. (C) Immunohistochemical staining for pimonidazole adducts in UUO kidneys of TbRIIendo+/+ and TbRIIendo+/2 mice. Original magnification, 310 and 340.
identify myofibroblasts of endothelial origin, we performed double staining for a-SMA and CD31 in kidney sections of TbRIIendo+/+ and TbRIIendo+/2 mice with and without FAN and in the UUO mice. Figure 8A shows representative images of a-SMA/CD31 double-positive cells in TbRIIendo+/+ mice treated with FA, which was significantly reduced in similarly treated TbRIIendo+/2 mice. Figure 9A shows representative images of kidneys from UUO mice of both groups again attesting to the reduced numbers of a-SMA/CD31 double-positive cells J Am Soc Nephrol 26: ccc–ccc, 2014
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in injured kidneys of TbRIIendo+/2 mice. Percentages of a-SMA/CD31 double-positive cells among CD31-positive ECs were quantified to be 22.7% in FA-treated TbRIIendo+/+ mice and 10.1% in TbRIIendo+/2 mice (n=5; P,0.01) (Figure 8B). These disparate numbers of EC conversion into myofibroblasts are consistent with the curtailed TGF-b signaling in TbRIIendo+/2 mice and further support the notion that TGF-b is an ultimate trigger to induce EndoMT.22 To obtain more rigorous evidence that the above “snapshot” figures of endothelialto-mesenchyme transiting cells faithfully reflect the proportion of myofibroblasts originating from ECs in TbRIIendo+/2 mice, we costained kidney sections from mice with and without FA treatment for Cre-recombinase and a-SMA. Expression of Cre-transgene, as a stable marker during epithelial-to-mesenchymal transition in kidney fibrosis was recently demonstrated.23 This lineage tracing approach is valuable in our study because of its ability to positively identify ECs (because Cre-recombinase expression in TbRIIendo+/2 mice is driven by the Tie2 promoter). In addition, it circumvents problems arising from the potential loss of endogenous endothelial markers, such as CD31, during EndoMT. Findings presented in Figure 10 demonstrate that the percentages of a-SMA/Cre double-positive cells among Cre-positive ECs were comparable with percentages of a-SMA/CD31 double-positive cells among CD31-positive ECs (Figure 8B). Moreover, in cultured renal ECs, staining for a-SMA–positive cells after TGF-b1 treatment was significantly reduced in TbRII+/2 ECs (Figure 11C). These data demonstrate that EndoMT is suppressed approximately 2.0- to 2.5-fold in TbRIIendo+/2 mice, a finding that may underlie the improved patency of renal microvasculature and reduced fibrotic response in the FAN model.
DISCUSSION
The data presented herein are based on a novel mouse model of curtailed TGF-b signaling in ECs, which is characterized by a normal phenotype under unstressed conditions, but exhibits a remarkable protection against fibrotic complications of FAN and UUO. This protection is related to the reduced level of endothelial transition toward the mesenchymal phenotype. Endothelial TbRII Deficiency Blunts Fibrosis
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activation.26–28 The main switch between these two pathways is provided by endoglin,7,29,30 which itself is thought to be under regulatory control by TGF-b.31,32 Under basal conditions, balance between both pathways exist through endoglin inhibition of Smad3 signaling and activation of Smad1.33 However, it is unknown whether prolonged and intense TGF-b signaling in ECs is associated with the predominant switch to ALK5 pathway. For this reason, attempts at manipulating endothelial TGF-b signaling under stress situations have ample rationale, as detailed below. In chronic kidney injury, in contrast with acute renal injury, tubulointerstitial fibrosis is ameliorated when endothelial TGF-b signaling is curtailed. Distinct and opposing roles for TbRII in regulating renal fibrosis and inflammation were previously observed in mice with deletion of TbRII in proximal tubular epithelial cells,17 in accord with our observations in mice with endothelial TbRII impaired signaling. Of note, it is important to distinguish the effect of short- and longterm upregulation of TGF-b on cell biology, as exemplified by exhaustion of endoglin regulation of the ALK switch34 or by a switch from the L-endoglin isoform to the S-endoglin isoform, as shown in our study. Recognition of the role played by EndoMT in kidney fibrosis has grown in recent years. Our model provides an important tool to test this emerging notion. Our comparative analysis of the total number of CD31-labeled microvessels with the actual number of Figure 8. EndoMT in mice with FA nephrotoxicity. (A) Representative images for CD31 patent perfused microvessels detected by an (green) and a-SMA (red) staining (n=5). Wild-type control kidneys shows costaining for intravenously injected endothelial-specific CD31 and a-SMA only in vessels. These areas were excluded during quantitative analysis. (B) Quantitative analysis of CD31 plus a-SMA double-positive cells in lectin discloses a striking difference. It apTbRIIendo+/+ or TbRIIendo+/2 vehicle- or FA-treated mice. *P,0.01; #P,0.01. Original pears that in the kidney undergoing fibrosis, the total number of CD31-labeled vessels is magnification, 360. an inaccurate indicator of the microvascular compromise. The decline in lectin-labeled microvessels much exceeds the marginal changes in CD31 During progression of CKD, the most common finding in labeling, indicating that the patency of circulatory beds is both animals and humans is upregulation of TGF-b1.2,3,24 Its severely impaired with the lesser structural damage to the signaling is usually profibrotic, but is not without exceptions. endothelial lining. These findings are supported by the fact For instance, although conditional deletion of TbRII in proxthat we observed changes in perfusion and hypoxia in injured imal tubular epithelial cells inhibits tubulointerstitial fibrosis kidneys (Figure 7). Moreover, as shown in Figures 8 and 9, in kidneys with UUO,19 the opposite effect is observed when many CD31-positive cells actually undergo EndoMT, thus deletion targets epithelia of the collecting duct.18 masking the deteriorating microcirculation. On the basis ECs represent a unique target for TGF-b because they are of these considerations, we propose that (1) measuring mithe predominant cell type expressing an alternative receptor, crovascular patency is a better test to detect abnormalities in ALK1.25 The downstream canonical ALK5 cascade of Smad 2/3 with its antiangiogenic program coexists with the ALK1-induced tissue perfusion than labeling CD31-positive cells, and (2) that the discrepancy between the total CD31-positive and Smad 1/5 and its proangiogenic program of transcriptional 8
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imply that EndoMT contributes to renal fibrosis after prolonged, but not transient, injury.37 The better preservation of functional capillaries and improved angiogenic responses during kidney injury in our mouse model with curtailed endothelial TGF-b signaling represent additional evidence of subverted EndoMT and protection of these mice from developing fibrosis. The data collectively document that partial ablation of TbRII in the endothelium reduces fibrosis during chronic kidney injury. Our results indicate the critical role of excessive endothelial TGF-b signaling in enhanced EndoMT, defective angiogenic response to injury, and development of fibrosis.
CONCISE METHODS Animals
Figure 9. EndoMT in UUO mice. (A) Representative images for CD31 (green) and a-SMA (red) staining (n=4). No primary antibody controls from CD31 staining and a-SMA staining are also shown. (B) Quantitative analysis of CD31 plus a-SMA doublepositive cells in UUO kidneys of TbRIIendo+/+ and TbRIIendo+/2 mice. *P,0.01. Original magnification, 360.
lectin-positive microvessels is a direct consequence and measure of EndoMT. The source of myofibroblasts in fibrotic kidneys has been a subject of intense interest. Studies have suggested that fibrocytes, local fibroblasts, epithelial cells, or vascular pericytes are a major source of myofibroblasts.12–16 Each of these sources has experimental limitations due to the lack of specific markers. In light of these issues, our observations that curtailing endothelial TGF-b signaling is sufficient to blunt progressive scarring of injured kidneys is supported by the fact that myofibroblasts can be derived from the capillary endothelium through EndoMT.16,35 Participation of EndoMT in renal fibrosis has been shown in three mouse models of CKD (including Alport’s syndrome, diabetic nephropathy, and chronic nitric oxide synthase inhibition16,35,36) and our findings add to this list that mice with curtailed endothelial TGF-b signaling have significantly blunted EndoMT during chronic renal injury. Data also J Am Soc Nephrol 26: ccc–ccc, 2014
Tie2-Cre mice were obtained from The Jackson Laboratory [B6.Cg-Tg(Tek-cre)1Ywa/J]. TbRIIflox/ flox mice [strain name B6.129S6-Tgfbr2tm1Him], which have exon2 of TbRII flanked with lox-p sites as described by Chytil et al.,38 were obtained from the National Cancer Institute Mouse Repository in Maryland. All animal protocols were conducted in accordance with the National Institutes of Health (NIH) 1999 guidance and were approved by the Institutional Animal Care and Use Committee. Mice aged between 8 and 12 weeks were used in this study.
Genotyping
Genotyping was performed by tail DNA PCR analysis. Tail DNA was isolated using the REDExtract-N-Amp Tissue PCR Kit (SigmaAldrich, St. Louis, MO). Primer sequences used for genotyping floxed TbRII allele were as follows: TbRII forward, 59TAA ACA AGG TCC GGA GCC CA39; and TbRII reverse, 59ACT TCT GCA AGA GGT CCC CT39. Tie2 transgene was detected using the following: forward, 59GCG GTC TGG CAG TAA AAA CTA TC39; and reverse, 59 GTG AAA CAG CAT TGC TGT CAC TT39. PCR products were analyzed with acrylamide gels.
Aortic Endothelial Sprouting Assay Thoracic aortas were obtained from 12-week-old mice. Aortic rings sectioned with 1-mm intervals were embedded in three-dimensional matrigel in culture chambers. Newly formed capillary cords in explant cultures were imaged for 6–13 days. The number of capillary sprouts along the perimeter of each explant was counted by two independent observers blinded to the origin of explant cultures. Explants were imaged using an inverted fluorescence microscope (Nikon) equipped with a charge-coupled device camera (Hamamatsu Photonics). Endothelial TbRII Deficiency Blunts Fibrosis
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with a creatinine assay kit (Cayman Chemicals, Ann Arbor, MI). Proteinuria was expressed as the urinary albumin/creatinine ratio. Serum creatinine concentration was measured using a commercially available kit (Abcam, Inc., MA).
Isolation of Primary Renal ECs ECs were isolated from kidneys dissected from 12-week-old TbRIIendo+/+ or TbRIIendo+/2 mice, according to the previously published protocol.41 Perfused kidneys were diced and tissue fragments were then digested with Dispase II (0.8 U/ml; Roche, Mannheim) and Collagenase H (1 mg/ml; Roche) in DMEM/F12 medium containing 2% FBS and antibiotics at 37°C for 1 hour, after which the suspension was homogenized by trituration. The homogenate was filtered through 100-mm nylon mesh (Falcon; BD Biosciences, San Jose, CA) followed by 40-mm nylon mesh and pelleted by centrifugation (300g for 6 minutes). Cells were resuspended in 1% BSA in PBS and incubated with anti-rat Dynabeads (Invitrogen, Carlsbad, CA) coated with rat anti-mouse CD31 (BD Biosciences Pharmingen, endo+/2 Figure 10. EndoMT in TbRII mice with FA nephrotoxicity. (A) Representative imSan Diego, CA) at 4°C for 20 minutes, washed, ages for Cre (red) and a-SMA (green) staining (n=5). Arrows indicate a-SMA–stained areas with and without Cre costaining. (B) Quantitative analysis of Cre plus a-SMA double-positive magnetically separated and ECs were cultured on gelatin-coated plates in Iscove’s modified cells in TbRIIendo+/2 vehicle or FA-treated mice. *P,0.01. Original magnification, 340. Dulbecco’s medium containing 15% FBS, 1% BSA, 0.01 mg/ml recombinant human insulin, 0.2% mg/ml human Quantitative angiogenesis assays were performed according to pretransferrin, 100 ng/ml each of recombinant mouse vascular endothelial viously published protocols.39,40 growth factor, basic fibroblast growth factor, and epidermal growth factor (all from Life Technologies, Guilford, CT) at 37°C in a humidified Folate Model of Acute Renal Injury and Chronic 5% CO2 atmosphere. Cells were serum starved overnight and treated Nephropathy either with recombinant TGF-b1 or TGF-b2 (5 ng/ml) (R&D Systems, A single intraperitoneal injection of FA (250 mg/g body weight) was Minneapolis, MN) for 30 minutes for protein and 48 hours for RNA administered in 12-week-old mice, and kidneys and whole blood colisolation. lected from FA-treated or vehicle-treated animals. Mice were injected with FA and euthanized at 48 hours (AKI phase) or at 6 weeks (chronic fibrotic phase). Kidneys were fixed in 4% paraformaldehyde and emQuantitative PCR Analyses Total RNA was isolated using a Spinsmart RNA Mini purification kit bedded in paraffin. Paraffin sections (4-mm thick) were stained with from Denville Scientific Inc. (Metuchen, NJ). One microgram of RNA hematoxylin and eosin and periodic acid–Schiff and were examined by a was reverse transcribed using the High-Capacity RNA-cDNA kit nephropathologist blinded to the origin of individual preparations. Par(Applied Biosystems, Foster City, CA). Real-time quantitative PCR affin sections were also stained with Masson’s trichrome and fibrosis was performed using Perfecta SYBR Green FastMix on a Stratagene was quantified using the NIH ImageJ program. MX3000P. The following primers were used: COL1 forward primer, 59CTG CTG GCA AAG ATG GAG A39; COL1 reverse primer, 59ACC UUO AGG AAG ACC CTG GAA TC39; COL3 forward primer, 59 CAA ATG Mice were anesthetized with isoflurane and placed in the left lateral GCATCC CAG GAG39; COL3 reverse primer, 59CAT CTC GGC CAG decubitus position. The left ureter was identified and ligated at the level of GTT CTC39; TbRII forward primer, 59GTG AGA AGC CGC ATG the lower pole of the kidney with 3-0 silk sutures through a left flank AAG TC39; TbRII reverse primer, 59GGG ACT GCT GGT GGT GTA incision. Mice were euthanized after 2 weeks and kidneys were processed TT39; S-ENDO forward primer, 59-TGA GTA TCC CAA GCC TCC as described above. Contralateral kidneys were used as controls. AGC CCA T-39; S-ENDO reverse primer, 59-CTG AGG GGC GTG GGT GAA GGT CAG; L-ENDO forward primer, 59-GCA CTC TGG Microalbuminuria, Serum Creatinine, and BP TAC ATC TAT TCT CAC ACA CGT GG-39; and L-ENDO reverse Measurements primer, 59-GGG CAC TAC GCC ATG CTG CTG GTG G-39. Values Urine albumin content was measured using mouse albumin-specific were normalized for the abundance of the amplified 18s rRNA in each ELISA (Exocell Laboratories, PA) and creatinine was determined 10
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Immunofluorescence Staining
Kidneys were fixed in 4% paraformaldehyde overnight, transferred to PBS containing 30% sucrose overnight, embedded in O.C.T. (TissueTek; Sakura Finetek, Inc., Torrance, CA), and cryosectioned (7-mm thick sections).44 Sections were blocked with PBS-BSA (1%) and stained with rat mAb to CD31 (PECAM-1; BD Biosciences Pharmingen) followed by AlexaFluor FITC-conjugated goat anti-rat (Invitrogen) secondary antibody, according to the manufacturer’s recommendation. Nuclei were stained with mounting fluid containing 49,6-diamidino-2phenylindole(Vector Laboratories, Burlingame, CA). Costaining of CD31 or Cre-recombinase and a-SMA on frozen sections was done by permeabilizing in 0.3% Triton3100 in PBS, blocking in PBS-BSA (1%) followed by staining with CD31 antibody or mouse mAb to Cre (SigmaAldrich) and rabbit polyclonal anti-a-SMA antibody (Abcam, Inc.) followed by AlexaFluor FITC–conjugated goat anti-rat or AlexaFluor +/+ +/2 Figure 11. Modulation of EndoMT in TbRII and TbRII kidney ECs. (A) Immu- 594–conjugated goat anti-mouse and Alexa+/+ and TbRII+/2 kidney Fluor 594– or AlexaFluor 488–conjugated goat nofluorescence images for CD31 or a-SMA in cultured TbRII ECs treated with TGF-b1 (5 ng/ml) for 6 days (n=5). The nuclei are stained with 49,6- anti-rabbit (Invitrogen) secondary antibodies. diamidino-2-phenylindole. (B) Quantitative analysis of a-SMA–positive cells per 100 Primary ECs were cultured on gelatin-coated cells. *P,0.002. Original magnification, 310 for a-SMA; 340 for CD31 and a-SMA. eight-well culture slides (Lab-Tek Chamber Slides; Thermo Fisher Scientific, Vernon Hills, IL) and fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.3% Triton X-100 for 5 minutes, and experiment. Fold change in gene expression was determined using -DDCT 42 blocked with PBS/1% BSA for 1 hour. Cells were incubated with the 2 threshold cycle method as previously described. primary anti-CD31, anti-CD144 (VE-Cadherin; BD Biosciences Pharmingen), or anti-a-SMA antibody, followed by incubation Detection of Patent Vessels with secondary antibodies. Images were obtained using a compound To visualize patent vasculature mice were injected with 100 ml of Texas Nikon microscope (TE-2000U) equipped with a Spot digital camera Red-labeled L. esculentum lectin (1 mg/ml in 10 mm HEPES, 150 mm (Diagnostic Instruments, Inc., Sterling Heights, MI). Quantitative NaCl, pH 7.5; Sigma-Aldrich) via the tail vein 5 minutes before analysis of lectinand CD31-positive vessels was performed by meaeuthanasia. suring average lengths of capillaries using Metavue Software. Quantification of percentages of CD31-positive or Cre-positive and Laser-Doppler Flowmetry of Renal Blood Flow a-SMA–positive cells was performed using the grid method with Renal blood flow was evaluated 14 days after UUO under xylazine/ images taken under 340 or 360 magnification. Results are presented ketamine sedation. A midline incision was performed to access the as ratios of CD31 or Cre and a-SMA double-positive areas to CD31kidneys. Cortical and medullary renal blood flow was measured using or Cre-positive areas. the laser-Doppler Perfusion Monitoring probes, image scanner, and accompanying software (PeriScan PIM II, PeriFlux system 5000, and LDPIwin version 2.6.1, respectively; PeriMed Instruments, PA). The correct positioning of laser-Doppler probes was verified after completion of each experiment.
Detection of Pimonidazole Adducts To analyze renal oxygenation in UUO mice, mice received an intraperitoneal injection of 60 mg/kg pimonidazole HCL (Hypoxyprobe-1; Hypoxyprobe, Inc., Burlington, MA) 50 minutes before euthanasia. Pimonidazole immunostaining was performed using a Hypoxyprobe-1 Plus Kit (Hypoxyprobe, Inc.) on 4-mm thick paraffin sections.43 J Am Soc Nephrol 26: ccc–ccc, 2014
Western Blotting Cells were washed with ice-cold PBS and lysed in radioimmunoprecipitation assay buffer. Lysates were clarified by centrifugation, and protein concentration was determined by the BCA protein assay (Thermo Fisher Scientific). Proteins were separated on 4%–20% SDSPAGE gels (Bio-Rad) and transferred. Membranes were blocked and incubated with phospho Smad2, phospho Smad1/5, total Smad2, or total Smad1 antibodies (Cell Signaling Technologies, Danvers, MA) overnight at 4°C and secondary antibodies (GE Healthcare) for 1 hour at room temperature. The bands were visualized using a SuperSignal chemiluminescence kit (Thermo Fisher Scientific). Endothelial TbRII Deficiency Blunts Fibrosis
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Statistical Analyses All experiments were repeated at least three times. All values are expressed as the mean6SD. Data were analyzed using ANOVA with post hoc analysis for multiple group comparisons using Bonferroni method. P,0.05 was considered statistically significant.
ACKNOWLEDGMENTS These studies were supported by grants from the NIH (DK54602, DK052783, and DK45462) and Westchester Artificial Kidney Foundation (to M.S.G.), as well as an intramural grant from New York Medical College (to S.X.).
DISCLOSURES None.
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15. Lin SL, Kisseleva T, Brenner DA, Duffield JS: Pericytes and perivascular fibroblasts are the primary source of collagen-producing cells in obstructive fibrosis of the kidney. Am J Pathol 173: 1617– 1627, 2008 16. Zeisberg EM, Potenta SE, Sugimoto H, Zeisberg M, Kalluri R: Fibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transition. J Am Soc Nephrol 19: 2282–2287, 2008 17. Meng XM, Huang XR, Xiao J, Chen HY, Zhong X, Chung ACK, Lan HY: Diverse roles of TGF-b receptor II in renal fibrosis and inflammation in vivo and in vitro. J Pathol 227: 175–188, 2012 18. Gewin L, Bulus N, Mernaugh G, Moeckel G, Harris RC, Moses HL, Pozzi A, Zent R: TGF-beta receptor deletion in the renal collecting system exacerbates fibrosis. J Am Soc Nephrol 21: 1334–1343, 2010 19. Gewin L, Vadivelu S, Neelisetty S, Srichai MB, Paueksakon P, Pozzi A, Harris RC, Zent R: Deleting the TGF-b receptor attenuates acute proximal tubule injury. J Am Soc Nephrol 23: 2001–2011, 2012 20. Nicosia RF, Zhu WH, Fogel E, Howson KM, Aplin AC: A new ex vivo model to study venous angiogenesis and arterio-venous anastomosis formation. J Vasc Res 42: 111–119, 2005 21. Blanco FJ, Grande MT, Langa C, Oujo B, Velasco S, RodriguezBarbero A, Perez-Gomez E, Quintanilla M, López-Novoa JM, Bernabeu C: S-endoglin expression is induced in senescent endothelial cells and contributes to vascular pathology. Circ Res 103: 1383–1392, 2008 22. Arciniegas E, Sutton AB, Allen TD, Schor AM: Transforming growth factor beta 1 promotes the differentiation of endothelial cells into smooth muscle-like cells in vitro. J Cell Sci 103: 521–529, 1992 23. Simic P, Zainabadi K, Bell E, Sykes DB, Saez B, Lotinun S, Baron R, Scadden D, Schipani E, Guarente L: SIRT1 regulates differentiation of mesenchymal stem cells by deacetylating b-catenin. EMBO Mol Med 5: 430–440, 2013 24. Wynn TA: Cellular and molecular mechanisms of fibrosis. J Pathol 214: 199–210, 2008 25. Roelen BAJ, van Rooijen MA, Mummery CL: Expression of ALK-1, a type 1 serine/threonine kinase receptor, coincides with sites of vasculogenesis and angiogenesis in early mouse development. Dev Dyn 209: 418–430, 1997 26. Goumans MJ, Valdimarsdottir G, Itoh S, Lebrin F, Larsson J, Mummery C, Karlsson S, ten Dijke P: Activin receptor-like kinase (ALK)1 is an antagonistic mediator of lateral TGFbeta/ALK5 signaling. Mol Cell 12: 817–828, 2003 27. Goumans MJ, Valdimarsdottir G, Itoh S, Rosendahl A, Sideras P, ten Dijke P: Balancing the activation state of the endothelium via two distinct TGF-beta type I receptors. EMBO J 21: 1743–1753, 2002 28. Ray BN, Lee NY, How T, Blobe GC: ALK5 phosphorylation of the endoglin cytoplasmic domain regulates Smad1/5/8 signaling and endothelial cell migration. Carcinogenesis 31: 435–441, 2010 29. Blanco FJ, Bernabeu C: Alternative splicing in endothelial senescence: Role of the TGF-b co-receptor endoglin. In: Senescence, edited by Nagata T, Rijeka, Croatia, InTech, 2012, pp 499–518 30. Blanco FJ, Santibanez JF, Guerrero-Esteo M, Langa C, Vary CPH, Bernabeu C: Interaction and functional interplay between endoglin and ALK-1, two components of the endothelial transforming growth factorbeta receptor complex. J Cell Physiol 204: 574–584, 2005 31. Rodríguez-Peña A, Eleno N, Düwell A, Arévalo M, Pérez-Barriocanal F, Flores O, Docherty N, Bernabeu C, Letarte M, López-Novoa JM: Endoglin upregulation during experimental renal interstitial fibrosis in mice. Hypertension 40: 713–720, 2002 32. Ota T, Fujii M, Sugizaki T, Ishii M, Miyazawa K, Aburatani H, Miyazono K: Targets of transcriptional regulation by two distinct type I receptors for transforming growth factor-beta in human umbilical vein endothelial cells. J Cell Physiol 193: 299–318, 2002 33. Scherner O, Meurer SK, Tihaa L, Gressner AM, Weiskirchen R: Endoglin differentially modulates antagonistic transforming growth factor-beta1 and BMP-7 signaling. J Biol Chem 282: 13934–13943, 2007
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34. Gaengel K, Genové G, Armulik A, Betsholtz C: Endothelial-mural cell signaling in vascular development and angiogenesis. Arterioscler Thromb Vasc Biol 29: 630–638, 2009 35. Li J, Qu X, Bertram JF: Endothelial-myofibroblast transition contributes to the early development of diabetic renal interstitial fibrosis in streptozotocin-induced diabetic mice. Am J Pathol 175: 1380–1388, 2009 36. O’Riordan E, Mendelev N, Patschan S, Patschan D, Eskander J, CohenGould L, Chander P, Goligorsky MS: Chronic NOS inhibition actuates endothelial-mesenchymal transformation. Am J Physiol Heart Circ Physiol 292: H285–H294, 2007 37. Kanasaki K, Taduri G, Koya D: Diabetic nephropathy: The role of inflammation in fibroblast activation and kidney fibrosis. Front Endocrinol (Lausanne) 4: 7, 2013 38. Chytil A, Magnuson MA, Wright CV, Moses HL: Conditional inactivation of the TGF-beta type II receptor using Cre:Lox. Genesis 32: 73–75, 2002 39. Brodsky S, Chen J, Lee A, Akassoglou K, Norman J, Goligorsky MS: Plasmin-dependent and -independent effects of plasminogen activators and inhibitor-1 on ex vivo angiogenesis. Am J Physiol Heart Circ Physiol 281: H1784–H1792, 2001 40. Chen J, Brodsky S, Li H, Hampel DJ, Miyata T, Weinstein T, Gafter U, Norman JT, Fine LG, Goligorsky MS: Delayed branching of endothelial
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capillary-like cords in glycated collagen I is mediated by early induction of PAI-1. Am J Physiol Renal Physiol 281: F71–F80, 2001 Fang S, Wei J, Pentinmikko N, Leinonen H, Salven P: Generation of functional blood vessels from a single c-kit+ adult vascular endothelial stem cell. PLoS Biol 10: e1001407, 2012 Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25: 402–408, 2001 Stoessel A, Paliege A, Theilig F, Addabbo F, Ratliff B, Waschke J, Patschan D, Goligorsky MS, Bachmann S: Indolent course of tubulointerstitial disease in a mouse model of subpressor, low-dose nitric oxide synthase inhibition. Am J Physiol Renal Physiol 295: F717–F725, 2008 Chen J, Park HC, Addabbo F, Ni J, Pelger E, Li H, Plotkin M, Goligorsky MS: Kidney-derived mesenchymal stem cells contribute to vasculogenesis, angiogenesis and endothelial repair. Kidney Int 74: 879–889, 2008
This article contains supplemental material online at http://jasn.asnjournals. org/lookup/suppl/doi:10.1681/ASN.2013101137/-/DCSupplemental.
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Curtailing Endothelial TGF-b Signaling Is Sufficient to Reduce Endothelial-Mesenchymal Transition and Fibrosis in CKD Sandhya Xavier,*† Radovan Vasko,*†‡ Kei Matsumoto,*† Joseph A. Zullo,*† Robert Chen,*† Julien Maizel,*† Praveen N. Chander,§ and Michael S. Goligorsky*† Departments of *Medicine, Pharmacology, Physiology, and §Pathology, and †Renal Research Institute, New York Medical College, Valhalla, New York; and ‡Department of Nephrology and Rheumatology, University Medical Center, Goettingen, Germany
ABSTRACT Excessive TGF-b signaling in epithelial cells, pericytes, or fibroblasts has been implicated in CKD. This list has recently been joined by endothelial cells (ECs) undergoing mesenchymal transition. Although several studies focused on the effects of ablating epithelial or fibroblast TGF-b signaling on development of fibrosis, there is a lack of information on ablating TGF-b signaling in the endothelium because this ablation causes embryonic lethality. We generated endothelium-specific heterozygous TGF-b receptor knockout (TbRIIendo+/2) mice to explore whether curtailed TGF-b signaling significantly modifies nephrosclerosis. These mice developed normally, but showed enhanced angiogenic potential compared with TbRIIendo+/+ mice under basal conditions. After induction of folic acid nephropathy or unilateral ureteral obstruction, TbRIIendo+/2 mice exhibited less tubulointerstitial fibrosis, enhanced preservation of renal microvasculature, improvement in renal blood flow, and less tissue hypoxia than TbRIIendo+/+ counterparts. In addition, partial deletion of TbRII in the endothelium reduced endothelial-to-mesenchymal transition (EndoMT). TGF-b–induced canonical Smad2 signaling was reduced in TbRII+/2 ECs; however, activin receptor-like kinase 1 (ALK1)–mediated Smad1/5 phosphorylation in TbRII+/2 ECs remained unaffected. Furthermore, the S-endoglin/L-endoglin mRNA expression ratio was significantly lower in TbRII+/2 ECs compared with TbRII+/+ ECs. These observations support the hypothesis that EndoMT contributes to renal fibrosis and curtailing endothelial TGF-b signals favors Smad1/5 proangiogenic programs and dictates increased angiogenic responses. Our data implicate endothelial TGF-b signaling and EndoMT in regulating angiogenic and fibrotic responses to injury. J Am Soc Nephrol 26: ccc–ccc, 2014. doi: 10.1681/ASN.2013101137
CKD leading to end stage renal failure is associated with tissue scarring or fibrosis.1 For development of effective therapeutic strategies, it is essential to define the cellular and molecular mechanisms that modulate regeneration of kidney.2 There is strong evidence that TGF-b1 is a key mediator in the pathogenesis of renal fibrosis in both mouse models and human kidney diseases.1–3 Direct evidence for its role in renal fibrosis comes from studies demonstrating that mice overexpressing an active form of TGF-b1 in the liver develop both progressive liver and renal fibrosis.4 Although restoration of adequate microvascular supply is essential for renal regeneration J Am Soc Nephrol 26: ccc–ccc, 2014
after chronic injury, genetic studies have revealed the pivotal role of TGF-b signaling in angiogenesis, as well as vascular integrity.5 Incidentally, loss of
Received October 30, 2013. Accepted June 25, 2014. Published online ahead of print. Publication date available at www.jasn.org. Correspondence: Dr. Sandhya Xavier or Michael S. Goligorsky, Department of Medicine, Renal Research Institute, New York Medical College, 15 Dana Road, Valhalla, NY 10595. Email: [email protected] or [email protected] Copyright © 2014 by the American Society of Nephrology
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peritubular capillaries, a hallmark of progressive renal disease in humans, plays a role in the etiology of interstitial fibrosis and tubular atrophy.6 TGF-b signal is initiated when the ligand binds to its cognate TGF-b type II receptor (TbRII) leading to phosphorylation of TbRI (also known as ALK5). The activated TbRI kinase phosphorylates canonical Smad2/3, which translocates to the nucleus and controls gene expression to inhibit endothelial proliferation, migration, and capillary tube formation. 7 TGF-b can also bind to activin receptor-like kinase ALK1 exclusively in endothelial cells (ECs), which induces Smad1/5 to potentiate angiogenic programs. Phenotypic and molecular
Figure 1. Characterization of endothelial TbRII heterozygote mice. (A) Genotyping of pups from TGF-bRIIFlox/Flox and Tie2-Cre: TGF-bRIIFlox/WT matings. Tail PCR genotyping analysis. The top panel shows detection of floxed and WT fragments. The bottom panel shows detection of Cre-transgene. (B) Real-time PCR analysis for TbRII mRNA expression in kidney ECs isolated from TbRIIendo+/+ and TbRIIendo+/2 mice (n=4). *P,0.05, TbRII+/+ versus TbRII+/2. (C) Western blot for Smad2 signaling in kidney ECs. WT, wild type; Cont, control.
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characterization of knockout mice for TGF-b signaling components have demonstrated their critical role in angiogenesis and cardiovascular system.8 Mice with ablation of endothelial TbRII or TbRI (ALK5) show vascular defects in the yolk sac and embryonic lethality as early as embryonic day 10.5. The embryos also have severe anemia and defective vasculogenesis,9,10 both signs of impaired differentiation of hemangioblast to hematopoietic stem cells and endothelial progenitor cells.
Figure 2. Functional characterization of TbRII heterozygote mice. (A) Images and quantification of ex vivo angiogenesis assays in three-dimensional matrigel using explant cultures of aortic ring segments at day 6 from TbRIIendo+/+ and TbRIIendo+/2 mice (n=5). (B) Western blot for Smad1/5 signaling in kidney ECs isolated from TbRIIendo+/+ and TbRIIendo+/2 mice. (C) Endoglin S/L mRNA expression ratios in kidney ECs. *P,0.05, TbRII+/+ versus TbRII+/2 and TGF-b1 treated TbRII+/+ versus TbRII+/2. Cont, control.
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Nguyen et al.11 recently showed that deletion of TbRII in ECs results in impaired blood vessel morphogenesis in the brain and intracerebral hemorrhage leading to embryonic lethality. The consequences of the partial ablation of TbRII in ECs remain unexplored. Collectively, these studies confirm that TGF-b signaling in ECs is essential for precise regulation of blood vessel assembly and consequently, normal embryonic development. Although increasing evidence shows the role of TGF-b1 in vasculogenesis or renal inflammation and fibrosis, the role of EC TGF-b receptor signaling in the progression of kidney diseases remains unexplored. The link between endothelial dysfunction and progression of fibrosis awaits elucidation. Several cell types have been proposed as initiators of tubulointerstitial fibrosis, including tubular epithelial cells, pericytes, fibroblasts, and more recently ECs, all of which can undergo mesenchymal transition to form myofibroblasts.12–16
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Indeed, several studies focused on the ablation of epithelial TGF-b signaling or depletion of TbRII in kidney fibroblasts to alter the development of fibrosis.17 The same strategy has been reported for epithelial cells in the proximal tubule and collecting duct.18,19 However, there are no published data on its ablation in the endothelium partly because of the unavailability of endothelial TbRII knockout mice due to the embryonic lethality.9,10 To address the question of whether endothelial TGF-b signals contribute to renal fibrosis, we utilized mice with partial ablation of TbRII in this study (i.e., mice heterozygous for TbRII), and investigated the role of endothelial TGF-b signaling in renal fibrosis of the folic acid nephropathy (FAN) and unilateral ureteral obstruction (UUO) models. The data demonstrate that curtailing TGF-b signaling in vascular ECs is sufficient to reduce endothelial-tomesenchymal transition (EndoMT), improve angiogenesis, and reduce fibrosis.
Figure 3. Fibrosis is reduced in TbRIIendo+/2 mice with FA toxicity. (A) Representative images of Masson’s trichrome–stained kidney sections from 12-week-old TbRIIendo+/+ and TbRIIendo+/2 mice treated with vehicle or FA 6 weeks after injection (n=5). (B) Quantification of fibrotic area (color quantification method). (C) Expression of collagen I and collagen III examined by quantitative real-time PCR. *P,0.05, treated TbRIIendo+/+ versus treated TbRIIendo+/2. Original magnification, 34 and 310. J Am Soc Nephrol 26: ccc–ccc, 2014
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RESULTS Generation of Mice with Endothelial Deletion of TbRII
We intercrossed mice homozygous for the floxed TbRII allele with mice heterozygous for Cre-recombinase under the control of the Tie2-promoter to obtain Tie2-Cre;TbRIIflox/+, which were crossed with TbRIIflox/flox mice. Genotyped offspring from these
intercrosses identified heterozygotes (Tie2-Cre;TbRIIflox/+) and wild-type pups (TbRIIflox/flox or TbRIIflox/+), whereas homozygote pups (Tie2-Cre; TbRIIflox/flox) were embryonic lethal, as previously reported.9–11 Percentages of these genotypes were found to be 32.3% each for TbRIIflox/flox and TbRIIflox/+ mice and 35.5% of Tie2-Cre;TbRIIflox/+ mice. Figure 1A shows results of genotyping PCR analysis of mice. Heterozygous mice
Figure 4. Fibrosis is reduced in TbRIIendo+/2 UUO mice. (A) Representative images of Masson’s trichrome–stained UUO and contralateral kidney sections from TbRIIendo+/+ and TbRIIendo+/2 mice (n=4). (B) Quantification of fibrotic area (color quantification method) *P,0.01, TbRIIendo+/+ UUO versus TbRIIendo+/2 UUO. (C) Representative images for a-SMA staining in UUO and contralateral kidney sections from TbRIIendo+/+ and TbRIIendo+/2 UUO mice. Original magnification, 34 and 310.
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(Tie2-Cre;TbRIIflox/+ hereafter referred to as TbRIIendo+/2) were viable, did not exhibit any phenotypic differences compared with control mice (TbRIIendo+/+), and showed no abnormalities in renal function at 6 weeks of age. We evaluated TbRII expression levels in kidney ECs isolated from TbRIIendo+/+ and TbRIIendo+/2 mice by quantitative real-time PCR analysis. Figure 1B documents the expected significant reduction in mRNA expression of TbRII in isolated ECs from TbRIIendo+/2 mice. When treated with TGF-b1 or TGF-b2 at a concentration of 5 ng/ml for 30 minutes, phosphorylation of Smad2 was reduced in ECs from TbRIIendo+/2 mice (Figure 1C). To ensure endothelial origin of isolated ECs, we stained cells for the expression of endothelial markers and showed that .90% of cells expressed VE-cadherin (CD144) and platelet-EC adhesion molecules (CD31) (Supplemental Figure 1). These data provided essential information on mice with partial endothelial ablation of TbRII signaling, demonstrating their viability, as opposed to total ablation, and normal development under unstressed conditions. Partial Endothelial Ablation of TbRII Signaling Boosts Ex Vivo Angiogenic Potential and Favors L-Endoglin– Mediated Smad1/5 Signaling
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because it is purely tubulopathic and does not affect ECs. Serum creatinine levels and urinary albumin/creatinine ratio were significantly increased in both TbRIIendo+/+ and TbRIIendo+/2 mice 48 hours after folic acid (FA) injection (Supplemental Figure 2, B and C). Both groups of mice showed frequent attenuation and focal loss of brush borders, focal mild- to-moderate tubular dilation, simplification, and rare necrosis of tubular epithelium. Granular eosinophilic material was observed in tubular profiles suggesting necrotic debris and was more prominent in TbRIIendo+/2 group (Supplemental Figure 2A). In contrast with our observations in acute renal injury, during chronic phase of injury, interstitial fibrosis was significantly ameliorated in the TbRIIendo+/2 +FA group compared with the TbRIIendo+/+ +FA group (Figure 3A). Consistent with these findings, expression of mRNA encoding collagen I and III was markedly increased in the TbRIIendo+/+ +FA group, whereas upregulation of these genes was ameliorated in the TbRIIendo+/2 +FA group of mice (Figure 3C). In a UUO model, a similar observation of reduced fibrosis in TbRIIendo+/2 mice was made (Figure 4B). Furthermore, the medullary region of UUO kidneys from TbRIIendo+/+ mice showed striking fibrosis, which
We evaluated the angiogenic potential using an ex vivo aortic endothelial sprouting assay.20 Data demonstrated that thoracic aortic rings obtained from TbRIIendo+/2 mice had accelerated sprouting in three-dimensional matrigel assays, which became clearly detectable by day 6 (Figure 2A). To prove that it was attributable to TGF-b signaling, we next examined the Smad1/5 profile in ECs isolated from kidneys of TbRIIendo+/+ and TbRIIendo+/2 mice, because ALK1-induced Smad1/5 signaling is proangiogenic, as opposed to the Smad2/3 pathway, which inhibits proliferation and migration of ECs. Smad1/5 levels at baseline were slightly reduced in ECs from TbRIIendo+/2 mice; nevertheless, after TGF-b1 or TGF-b2 treatment, phosphorylation of Smad1/5 was not impaired in ECs from TbRIIendo+/2 mice (Figure 2B). The expression ratio of short/long (S/L) isoforms of the auxiliary TGF-b1 receptor endoglin was significantly lower in ECs from TbRIIendo+/2 mice at baseline and after TGF-b1 treatment (Figure 2C). An increase in the S/L ratio of endoglin is known to be associated with senescence of ECs.21 Our observation of a reduced S/L ratio in ECs from TbRIIendo+/2 mice suggests that L-endoglin–mediated Smad1/5 proangiogenic signaling is more pronounced and increases angiogenic response, as seen ex vivo in TbRIIendo+/2 mice. This difference in angiogenic competence between TbRIIendo+/2 and TbRIIendo+/+ mice could play a role in vivo after imposition of stress, as was examined in the next series of experiments. Impaired Endothelial TbRII Signaling Improves Angiogenesis and Ameliorates Fibrotic Response in Chronic Kidney Injury
The FAN model was used to study the effects of acute and chronic injury in mice with partial deletion of TbRII in the endothelium. This model is advantageous for the study, J Am Soc Nephrol 26: ccc–ccc, 2014
Figure 5. Ex vivo angiogenesis assays in mice with FA nephrotoxicity. (A) Representative images of sprouting capillary cords in aortic explants at day 13. (B) Quantitative angiogenesis analysis in TbRIIendo+/+ or TbRIIendo+/2 vehicle or FA-treated animals. *P,0.01 untreated versus treated TbRIIendo+/+ or untreated versus treated TbRIIendo+/2.
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was ameliorated in the TbRIIendo+/2 group (Figure 4A). Consistent with this, a-smooth muscle actin (a-SMA) staining in the medulla was much more enhanced in UUO kidneys from
TbRIIendo+/+ mice compared with TbRIIendo+/2 mice (Figure 4C). Thoracic aortas were obtained from FAN mice to study their ex vivo angiogenic potential. Results showed that vessels obtained from mice heterozygous for endothelial TbRII have better angiogenic potential when challenged with FA compared with TbRIIendo+/+ mice, in which angiogenesis was found to be inhibited during FAinduced renal fibrosis (Figure 5). The impaired angiogenic response in TbRIIendo+/+ mice with FAN was associated with a significant loss of patent and functional capillaries in the kidney as judged by capillary density measurement using intravitally injected Lycopersicon esculentum (lectin) to label functional vessels, compared with CD31-positive total microvasculature in the kidneys (Figure 6A). Although CD31 staining was comparable in all groups of mice, lectin reporting only functional microvessels showed a dramatic decline in TbRIIendo+/+ mice with FAN, but TbRIIendo+/2 mice with FAN had a much higher proportion of functional microvessels. The mismatch between patent and total vessels also suggests initiation of an active EndoMT program after renal injury and that curtailed TGF-b signaling in the endothelium may restrain EndoMT transition, as addressed in the next series of experiments. In order to investigate the mechanisms underlying loss of patency of microvessels and to see whether this causes hypoperfusion and consequently tissue hypoxia, we performed laser-Doppler flowmetry of renal blood flow in the cortical and medullary regions of UUO kidneys. As shown in Figure 7, renal blood flow was impaired in the medulla of TbRIIendo+/+ mice, whereas flow in TbRIIendo+/2 mice was significantly preserved. We also probed kidneys for formation of pimonidazole adducts to measure regions with ,10 mmHg pO2 as represented in Figure 7C. A significant difference in staining was observed between both groups of mice with TbRIIendo+/+ mice displaying higher hypoxic regions compared with Figure 6. Microvascular density (CD31) and patency (lectin) in mice with FA neph- TbRIIendo+/2 mice.
rotoxicity. (A) Representative images for CD31 (green) and lectin (red) staining (n=5). (B) Average lengths (in micrometers) of CD31- or lectin-positive peritubular capillaries per image. (C) Ratios of average lengths of lectin- to CD31-positive capillaries (percentage) per image. *P,0.01, treated TbRIIendo+/+ versus TbRIIendo+/2 lectin-positive capillary length; **P,0.001, control versus FA-treated TbRIIendo+/+ mice; #P,0.01, treated TbRIIendo+/+ versus TbRIIendo+/2; N.S., P=NS for control versus FA-treated TbRIIendo+/2 mice. Original magnification, 340. Per the journal style, P values were rounded to two decimal places.
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Curtailed Endothelial TbRII Signaling Significantly Reduces EndoMT in FAN and UUO Injury
We investigated whether impaired TGF-b receptor signaling in the endothelium affects EndoMT processes during renal fibrosis. To J Am Soc Nephrol 26: ccc–ccc, 2014
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Figure 7. Renal blood flow measurement and pimonidazole staining in kidneys of TbRIIendo+/+ and TbRIIendo+/2 UUO mice. (A) Representative image scans from laserDoppler flowmetry analysis of contralateral and UUO kidneys from TbRIIendo+/+ and TbRIIendo+/2 mice (n=3). (B) Quantification of cortex and medullary renal blood flow in kidneys. *P,0.001, TbRIIendo+/+ and TbRIIendo+/2 UUO mice. (C) Immunohistochemical staining for pimonidazole adducts in UUO kidneys of TbRIIendo+/+ and TbRIIendo+/2 mice. Original magnification, 310 and 340.
identify myofibroblasts of endothelial origin, we performed double staining for a-SMA and CD31 in kidney sections of TbRIIendo+/+ and TbRIIendo+/2 mice with and without FAN and in the UUO mice. Figure 8A shows representative images of a-SMA/CD31 double-positive cells in TbRIIendo+/+ mice treated with FA, which was significantly reduced in similarly treated TbRIIendo+/2 mice. Figure 9A shows representative images of kidneys from UUO mice of both groups again attesting to the reduced numbers of a-SMA/CD31 double-positive cells J Am Soc Nephrol 26: ccc–ccc, 2014
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in injured kidneys of TbRIIendo+/2 mice. Percentages of a-SMA/CD31 double-positive cells among CD31-positive ECs were quantified to be 22.7% in FA-treated TbRIIendo+/+ mice and 10.1% in TbRIIendo+/2 mice (n=5; P,0.01) (Figure 8B). These disparate numbers of EC conversion into myofibroblasts are consistent with the curtailed TGF-b signaling in TbRIIendo+/2 mice and further support the notion that TGF-b is an ultimate trigger to induce EndoMT.22 To obtain more rigorous evidence that the above “snapshot” figures of endothelialto-mesenchyme transiting cells faithfully reflect the proportion of myofibroblasts originating from ECs in TbRIIendo+/2 mice, we costained kidney sections from mice with and without FA treatment for Cre-recombinase and a-SMA. Expression of Cre-transgene, as a stable marker during epithelial-to-mesenchymal transition in kidney fibrosis was recently demonstrated.23 This lineage tracing approach is valuable in our study because of its ability to positively identify ECs (because Cre-recombinase expression in TbRIIendo+/2 mice is driven by the Tie2 promoter). In addition, it circumvents problems arising from the potential loss of endogenous endothelial markers, such as CD31, during EndoMT. Findings presented in Figure 10 demonstrate that the percentages of a-SMA/Cre double-positive cells among Cre-positive ECs were comparable with percentages of a-SMA/CD31 double-positive cells among CD31-positive ECs (Figure 8B). Moreover, in cultured renal ECs, staining for a-SMA–positive cells after TGF-b1 treatment was significantly reduced in TbRII+/2 ECs (Figure 11C). These data demonstrate that EndoMT is suppressed approximately 2.0- to 2.5-fold in TbRIIendo+/2 mice, a finding that may underlie the improved patency of renal microvasculature and reduced fibrotic response in the FAN model.
DISCUSSION
The data presented herein are based on a novel mouse model of curtailed TGF-b signaling in ECs, which is characterized by a normal phenotype under unstressed conditions, but exhibits a remarkable protection against fibrotic complications of FAN and UUO. This protection is related to the reduced level of endothelial transition toward the mesenchymal phenotype. Endothelial TbRII Deficiency Blunts Fibrosis
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activation.26–28 The main switch between these two pathways is provided by endoglin,7,29,30 which itself is thought to be under regulatory control by TGF-b.31,32 Under basal conditions, balance between both pathways exist through endoglin inhibition of Smad3 signaling and activation of Smad1.33 However, it is unknown whether prolonged and intense TGF-b signaling in ECs is associated with the predominant switch to ALK5 pathway. For this reason, attempts at manipulating endothelial TGF-b signaling under stress situations have ample rationale, as detailed below. In chronic kidney injury, in contrast with acute renal injury, tubulointerstitial fibrosis is ameliorated when endothelial TGF-b signaling is curtailed. Distinct and opposing roles for TbRII in regulating renal fibrosis and inflammation were previously observed in mice with deletion of TbRII in proximal tubular epithelial cells,17 in accord with our observations in mice with endothelial TbRII impaired signaling. Of note, it is important to distinguish the effect of short- and longterm upregulation of TGF-b on cell biology, as exemplified by exhaustion of endoglin regulation of the ALK switch34 or by a switch from the L-endoglin isoform to the S-endoglin isoform, as shown in our study. Recognition of the role played by EndoMT in kidney fibrosis has grown in recent years. Our model provides an important tool to test this emerging notion. Our comparative analysis of the total number of CD31-labeled microvessels with the actual number of Figure 8. EndoMT in mice with FA nephrotoxicity. (A) Representative images for CD31 patent perfused microvessels detected by an (green) and a-SMA (red) staining (n=5). Wild-type control kidneys shows costaining for intravenously injected endothelial-specific CD31 and a-SMA only in vessels. These areas were excluded during quantitative analysis. (B) Quantitative analysis of CD31 plus a-SMA double-positive cells in lectin discloses a striking difference. It apTbRIIendo+/+ or TbRIIendo+/2 vehicle- or FA-treated mice. *P,0.01; #P,0.01. Original pears that in the kidney undergoing fibrosis, the total number of CD31-labeled vessels is magnification, 360. an inaccurate indicator of the microvascular compromise. The decline in lectin-labeled microvessels much exceeds the marginal changes in CD31 During progression of CKD, the most common finding in labeling, indicating that the patency of circulatory beds is both animals and humans is upregulation of TGF-b1.2,3,24 Its severely impaired with the lesser structural damage to the signaling is usually profibrotic, but is not without exceptions. endothelial lining. These findings are supported by the fact For instance, although conditional deletion of TbRII in proxthat we observed changes in perfusion and hypoxia in injured imal tubular epithelial cells inhibits tubulointerstitial fibrosis kidneys (Figure 7). Moreover, as shown in Figures 8 and 9, in kidneys with UUO,19 the opposite effect is observed when many CD31-positive cells actually undergo EndoMT, thus deletion targets epithelia of the collecting duct.18 masking the deteriorating microcirculation. On the basis ECs represent a unique target for TGF-b because they are of these considerations, we propose that (1) measuring mithe predominant cell type expressing an alternative receptor, crovascular patency is a better test to detect abnormalities in ALK1.25 The downstream canonical ALK5 cascade of Smad 2/3 with its antiangiogenic program coexists with the ALK1-induced tissue perfusion than labeling CD31-positive cells, and (2) that the discrepancy between the total CD31-positive and Smad 1/5 and its proangiogenic program of transcriptional 8
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imply that EndoMT contributes to renal fibrosis after prolonged, but not transient, injury.37 The better preservation of functional capillaries and improved angiogenic responses during kidney injury in our mouse model with curtailed endothelial TGF-b signaling represent additional evidence of subverted EndoMT and protection of these mice from developing fibrosis. The data collectively document that partial ablation of TbRII in the endothelium reduces fibrosis during chronic kidney injury. Our results indicate the critical role of excessive endothelial TGF-b signaling in enhanced EndoMT, defective angiogenic response to injury, and development of fibrosis.
CONCISE METHODS Animals
Figure 9. EndoMT in UUO mice. (A) Representative images for CD31 (green) and a-SMA (red) staining (n=4). No primary antibody controls from CD31 staining and a-SMA staining are also shown. (B) Quantitative analysis of CD31 plus a-SMA doublepositive cells in UUO kidneys of TbRIIendo+/+ and TbRIIendo+/2 mice. *P,0.01. Original magnification, 360.
lectin-positive microvessels is a direct consequence and measure of EndoMT. The source of myofibroblasts in fibrotic kidneys has been a subject of intense interest. Studies have suggested that fibrocytes, local fibroblasts, epithelial cells, or vascular pericytes are a major source of myofibroblasts.12–16 Each of these sources has experimental limitations due to the lack of specific markers. In light of these issues, our observations that curtailing endothelial TGF-b signaling is sufficient to blunt progressive scarring of injured kidneys is supported by the fact that myofibroblasts can be derived from the capillary endothelium through EndoMT.16,35 Participation of EndoMT in renal fibrosis has been shown in three mouse models of CKD (including Alport’s syndrome, diabetic nephropathy, and chronic nitric oxide synthase inhibition16,35,36) and our findings add to this list that mice with curtailed endothelial TGF-b signaling have significantly blunted EndoMT during chronic renal injury. Data also J Am Soc Nephrol 26: ccc–ccc, 2014
Tie2-Cre mice were obtained from The Jackson Laboratory [B6.Cg-Tg(Tek-cre)1Ywa/J]. TbRIIflox/ flox mice [strain name B6.129S6-Tgfbr2tm1Him], which have exon2 of TbRII flanked with lox-p sites as described by Chytil et al.,38 were obtained from the National Cancer Institute Mouse Repository in Maryland. All animal protocols were conducted in accordance with the National Institutes of Health (NIH) 1999 guidance and were approved by the Institutional Animal Care and Use Committee. Mice aged between 8 and 12 weeks were used in this study.
Genotyping
Genotyping was performed by tail DNA PCR analysis. Tail DNA was isolated using the REDExtract-N-Amp Tissue PCR Kit (SigmaAldrich, St. Louis, MO). Primer sequences used for genotyping floxed TbRII allele were as follows: TbRII forward, 59TAA ACA AGG TCC GGA GCC CA39; and TbRII reverse, 59ACT TCT GCA AGA GGT CCC CT39. Tie2 transgene was detected using the following: forward, 59GCG GTC TGG CAG TAA AAA CTA TC39; and reverse, 59 GTG AAA CAG CAT TGC TGT CAC TT39. PCR products were analyzed with acrylamide gels.
Aortic Endothelial Sprouting Assay Thoracic aortas were obtained from 12-week-old mice. Aortic rings sectioned with 1-mm intervals were embedded in three-dimensional matrigel in culture chambers. Newly formed capillary cords in explant cultures were imaged for 6–13 days. The number of capillary sprouts along the perimeter of each explant was counted by two independent observers blinded to the origin of explant cultures. Explants were imaged using an inverted fluorescence microscope (Nikon) equipped with a charge-coupled device camera (Hamamatsu Photonics). Endothelial TbRII Deficiency Blunts Fibrosis
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with a creatinine assay kit (Cayman Chemicals, Ann Arbor, MI). Proteinuria was expressed as the urinary albumin/creatinine ratio. Serum creatinine concentration was measured using a commercially available kit (Abcam, Inc., MA).
Isolation of Primary Renal ECs ECs were isolated from kidneys dissected from 12-week-old TbRIIendo+/+ or TbRIIendo+/2 mice, according to the previously published protocol.41 Perfused kidneys were diced and tissue fragments were then digested with Dispase II (0.8 U/ml; Roche, Mannheim) and Collagenase H (1 mg/ml; Roche) in DMEM/F12 medium containing 2% FBS and antibiotics at 37°C for 1 hour, after which the suspension was homogenized by trituration. The homogenate was filtered through 100-mm nylon mesh (Falcon; BD Biosciences, San Jose, CA) followed by 40-mm nylon mesh and pelleted by centrifugation (300g for 6 minutes). Cells were resuspended in 1% BSA in PBS and incubated with anti-rat Dynabeads (Invitrogen, Carlsbad, CA) coated with rat anti-mouse CD31 (BD Biosciences Pharmingen, endo+/2 Figure 10. EndoMT in TbRII mice with FA nephrotoxicity. (A) Representative imSan Diego, CA) at 4°C for 20 minutes, washed, ages for Cre (red) and a-SMA (green) staining (n=5). Arrows indicate a-SMA–stained areas with and without Cre costaining. (B) Quantitative analysis of Cre plus a-SMA double-positive magnetically separated and ECs were cultured on gelatin-coated plates in Iscove’s modified cells in TbRIIendo+/2 vehicle or FA-treated mice. *P,0.01. Original magnification, 340. Dulbecco’s medium containing 15% FBS, 1% BSA, 0.01 mg/ml recombinant human insulin, 0.2% mg/ml human Quantitative angiogenesis assays were performed according to pretransferrin, 100 ng/ml each of recombinant mouse vascular endothelial viously published protocols.39,40 growth factor, basic fibroblast growth factor, and epidermal growth factor (all from Life Technologies, Guilford, CT) at 37°C in a humidified Folate Model of Acute Renal Injury and Chronic 5% CO2 atmosphere. Cells were serum starved overnight and treated Nephropathy either with recombinant TGF-b1 or TGF-b2 (5 ng/ml) (R&D Systems, A single intraperitoneal injection of FA (250 mg/g body weight) was Minneapolis, MN) for 30 minutes for protein and 48 hours for RNA administered in 12-week-old mice, and kidneys and whole blood colisolation. lected from FA-treated or vehicle-treated animals. Mice were injected with FA and euthanized at 48 hours (AKI phase) or at 6 weeks (chronic fibrotic phase). Kidneys were fixed in 4% paraformaldehyde and emQuantitative PCR Analyses Total RNA was isolated using a Spinsmart RNA Mini purification kit bedded in paraffin. Paraffin sections (4-mm thick) were stained with from Denville Scientific Inc. (Metuchen, NJ). One microgram of RNA hematoxylin and eosin and periodic acid–Schiff and were examined by a was reverse transcribed using the High-Capacity RNA-cDNA kit nephropathologist blinded to the origin of individual preparations. Par(Applied Biosystems, Foster City, CA). Real-time quantitative PCR affin sections were also stained with Masson’s trichrome and fibrosis was performed using Perfecta SYBR Green FastMix on a Stratagene was quantified using the NIH ImageJ program. MX3000P. The following primers were used: COL1 forward primer, 59CTG CTG GCA AAG ATG GAG A39; COL1 reverse primer, 59ACC UUO AGG AAG ACC CTG GAA TC39; COL3 forward primer, 59 CAA ATG Mice were anesthetized with isoflurane and placed in the left lateral GCATCC CAG GAG39; COL3 reverse primer, 59CAT CTC GGC CAG decubitus position. The left ureter was identified and ligated at the level of GTT CTC39; TbRII forward primer, 59GTG AGA AGC CGC ATG the lower pole of the kidney with 3-0 silk sutures through a left flank AAG TC39; TbRII reverse primer, 59GGG ACT GCT GGT GGT GTA incision. Mice were euthanized after 2 weeks and kidneys were processed TT39; S-ENDO forward primer, 59-TGA GTA TCC CAA GCC TCC as described above. Contralateral kidneys were used as controls. AGC CCA T-39; S-ENDO reverse primer, 59-CTG AGG GGC GTG GGT GAA GGT CAG; L-ENDO forward primer, 59-GCA CTC TGG Microalbuminuria, Serum Creatinine, and BP TAC ATC TAT TCT CAC ACA CGT GG-39; and L-ENDO reverse Measurements primer, 59-GGG CAC TAC GCC ATG CTG CTG GTG G-39. Values Urine albumin content was measured using mouse albumin-specific were normalized for the abundance of the amplified 18s rRNA in each ELISA (Exocell Laboratories, PA) and creatinine was determined 10
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Immunofluorescence Staining
Kidneys were fixed in 4% paraformaldehyde overnight, transferred to PBS containing 30% sucrose overnight, embedded in O.C.T. (TissueTek; Sakura Finetek, Inc., Torrance, CA), and cryosectioned (7-mm thick sections).44 Sections were blocked with PBS-BSA (1%) and stained with rat mAb to CD31 (PECAM-1; BD Biosciences Pharmingen) followed by AlexaFluor FITC-conjugated goat anti-rat (Invitrogen) secondary antibody, according to the manufacturer’s recommendation. Nuclei were stained with mounting fluid containing 49,6-diamidino-2phenylindole(Vector Laboratories, Burlingame, CA). Costaining of CD31 or Cre-recombinase and a-SMA on frozen sections was done by permeabilizing in 0.3% Triton3100 in PBS, blocking in PBS-BSA (1%) followed by staining with CD31 antibody or mouse mAb to Cre (SigmaAldrich) and rabbit polyclonal anti-a-SMA antibody (Abcam, Inc.) followed by AlexaFluor FITC–conjugated goat anti-rat or AlexaFluor +/+ +/2 Figure 11. Modulation of EndoMT in TbRII and TbRII kidney ECs. (A) Immu- 594–conjugated goat anti-mouse and Alexa+/+ and TbRII+/2 kidney Fluor 594– or AlexaFluor 488–conjugated goat nofluorescence images for CD31 or a-SMA in cultured TbRII ECs treated with TGF-b1 (5 ng/ml) for 6 days (n=5). The nuclei are stained with 49,6- anti-rabbit (Invitrogen) secondary antibodies. diamidino-2-phenylindole. (B) Quantitative analysis of a-SMA–positive cells per 100 Primary ECs were cultured on gelatin-coated cells. *P,0.002. Original magnification, 310 for a-SMA; 340 for CD31 and a-SMA. eight-well culture slides (Lab-Tek Chamber Slides; Thermo Fisher Scientific, Vernon Hills, IL) and fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.3% Triton X-100 for 5 minutes, and experiment. Fold change in gene expression was determined using -DDCT 42 blocked with PBS/1% BSA for 1 hour. Cells were incubated with the 2 threshold cycle method as previously described. primary anti-CD31, anti-CD144 (VE-Cadherin; BD Biosciences Pharmingen), or anti-a-SMA antibody, followed by incubation Detection of Patent Vessels with secondary antibodies. Images were obtained using a compound To visualize patent vasculature mice were injected with 100 ml of Texas Nikon microscope (TE-2000U) equipped with a Spot digital camera Red-labeled L. esculentum lectin (1 mg/ml in 10 mm HEPES, 150 mm (Diagnostic Instruments, Inc., Sterling Heights, MI). Quantitative NaCl, pH 7.5; Sigma-Aldrich) via the tail vein 5 minutes before analysis of lectinand CD31-positive vessels was performed by meaeuthanasia. suring average lengths of capillaries using Metavue Software. Quantification of percentages of CD31-positive or Cre-positive and Laser-Doppler Flowmetry of Renal Blood Flow a-SMA–positive cells was performed using the grid method with Renal blood flow was evaluated 14 days after UUO under xylazine/ images taken under 340 or 360 magnification. Results are presented ketamine sedation. A midline incision was performed to access the as ratios of CD31 or Cre and a-SMA double-positive areas to CD31kidneys. Cortical and medullary renal blood flow was measured using or Cre-positive areas. the laser-Doppler Perfusion Monitoring probes, image scanner, and accompanying software (PeriScan PIM II, PeriFlux system 5000, and LDPIwin version 2.6.1, respectively; PeriMed Instruments, PA). The correct positioning of laser-Doppler probes was verified after completion of each experiment.
Detection of Pimonidazole Adducts To analyze renal oxygenation in UUO mice, mice received an intraperitoneal injection of 60 mg/kg pimonidazole HCL (Hypoxyprobe-1; Hypoxyprobe, Inc., Burlington, MA) 50 minutes before euthanasia. Pimonidazole immunostaining was performed using a Hypoxyprobe-1 Plus Kit (Hypoxyprobe, Inc.) on 4-mm thick paraffin sections.43 J Am Soc Nephrol 26: ccc–ccc, 2014
Western Blotting Cells were washed with ice-cold PBS and lysed in radioimmunoprecipitation assay buffer. Lysates were clarified by centrifugation, and protein concentration was determined by the BCA protein assay (Thermo Fisher Scientific). Proteins were separated on 4%–20% SDSPAGE gels (Bio-Rad) and transferred. Membranes were blocked and incubated with phospho Smad2, phospho Smad1/5, total Smad2, or total Smad1 antibodies (Cell Signaling Technologies, Danvers, MA) overnight at 4°C and secondary antibodies (GE Healthcare) for 1 hour at room temperature. The bands were visualized using a SuperSignal chemiluminescence kit (Thermo Fisher Scientific). Endothelial TbRII Deficiency Blunts Fibrosis
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Statistical Analyses All experiments were repeated at least three times. All values are expressed as the mean6SD. Data were analyzed using ANOVA with post hoc analysis for multiple group comparisons using Bonferroni method. P,0.05 was considered statistically significant.
ACKNOWLEDGMENTS These studies were supported by grants from the NIH (DK54602, DK052783, and DK45462) and Westchester Artificial Kidney Foundation (to M.S.G.), as well as an intramural grant from New York Medical College (to S.X.).
DISCLOSURES None.
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Endothelial TbRII Deficiency Blunts Fibrosis
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