Distinct sites of renal fibrosis in Crim1 mutant mice arise from multiple cellular origins
Crim1 is a transmembrane protein that regulates the bioavailability of growth factors such as VEGFA. Crim1KST264/KST264 hypomorphic mice develop renal disease characterized by glomerular cysts and loss of endothelial integrity, progressing to peritubular and pericystic fibrosis. Peritubular capillar...
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| Published in | The Journal of pathology Vol. 229; no. 5; pp. 685 - 696 |
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| Main Authors | , , , , |
| Format | Journal Article |
| Language | English |
| Published |
Chichester, UK
John Wiley & Sons, Ltd
01.04.2013
Wiley Subscription Services, Inc |
| Subjects | |
| Online Access | Get full text |
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| Summary: | Crim1 is a transmembrane protein that regulates the bioavailability of growth factors such as VEGFA. Crim1KST264/KST264 hypomorphic mice develop renal disease characterized by glomerular cysts and loss of endothelial integrity, progressing to peritubular and pericystic fibrosis. Peritubular capillary endothelial cells display morphological changes as well as detachment from the basement membrane. In this study, gene expression profiling of CD31+ endothelial cells isolated from Crim1KST264/KST264 kidneys showed up‐regulation of transcripts associated with fibrosis (Col3a1, Loxl1), endothelial dysfunction (Abp1, Dcn, Lcn2), biomarkers of renal damage (Lcn2, Havcr1/Kim1) as well as evidence for a TGFβ1/TNF‐associated inflammatory process. To determine whether the aberrant endothelium may in part contribute to the fibrogenic process, Tie2Cre‐DsRed lineage tracing was undertaken in Crim1KST264/KST264 mice. Approximately 31% of de novo αSMA+ myofibroblasts detected within the tubulointerstitium were Tie2+DsRed+. However, 5.3% were F4/80+DsRed+, indicating a small population of myofibroblasts of monocytic rather than endothelial origin. In contrast, only 12% of myofibroblasts located around glomerular cysts were Tie2+DsRed+, with 7.7% being monocyte‐derived (F4/80+DsRed+). Collectively, this model supports the involvement of endothelial cells/monocytes in fibrosis within the tubulointerstitium, but also the heterogeneity of the fibrotic process even within distinct regions of the same kidney. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. |
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| Bibliography: | ArticleID:PATH4155 List of primers used for quantitative PCRList of the top 200 B-statistics sorted genes from the microarray that were differentially expressed in the Crim1KST264/KST264 endothelial cells.Endothelial lineage tracing in the Crim1KST264/KST264 mice. The Tie2Cre and Z/Red mice were bred onto the Crim1+/KST264 mouse line and the respective males and females with the desired transgenes were selected for inter-crossing. Crim1+/+;Tie2;ZRed and Crim1KST264/KST264;Tie2;ZRed mice containing DsRed+ endothelial cells were selected for the lineage tracing studyIncreased Havcr1 protein within the Crim1KST264/KST264 renal endothelium. (A) The specificity of the Havcr1 antibody was first validated on wild-type mice that underwent unilateral ischaemia-reperfusion injury. Increased amount of Havcr1 was observed in the damaged kidney epithelial tubules but not in the contralateral kidney. (B) Co-immunofluorescence staining of the Crim1KST264/KST264 endothelium with tomato lectin showed increased Havcr1 localization within the fibrotic interstitium (double arrowheads) and endothelium layer (arrowheads) as compared to their wild-type counterparts. DAPI was used to identify nucleiTGFβ1 induced an endothelial-mesenchymal transition in the EOMA cell line. (A) Treatment of an immortalized endothelial cell line (EOMA) with TGFβ1 resulted in the up-regulation of several candidate genes that were present in the microarray gene list. (B) Phalloidin staining for actin filament also revealed the formation of stress fibres (arrowhead) in TGFβ1-treated EOMA. Additionally, a considerable reduction of CD31, an endothelial-specific marker, was observed in the treated EOMAValidation of Tie2Cre-DsRed expression in the renal endothelial cells. (A) In the absence of the Tie2Cre recombinase gene in Z/Red mice, no activation of DsRed fluorescence was observed. (B) Mice that inherited both copies of the Tie2Cre and Z/Red transgenes showed specific DsRed expression in the endothelium layer, as marked by CD31, an endothelial-specific marker (arrowheads). (C) FACS analysis showed that no detectable DsRed fluorescence was observed in the Z/Red endothelial population. (D) In the presence of the Tie2Cre recombinase transgene, 41.7% of the CD31 population was DsRed+. (E-G) Co-immunostaining of the TZWT and TZCrim1 kidneys with the monocyte F4/80 marker revealed that 21.7% and 22.5%, respectively, of the DsRed+ cells are monocytes. (H-J) In contrast, 34.7% and 59.6% of the DsRed+ population surrounding the TZWT and TZCrim1 glomerular regions, respectively, were F4/80+. Scale bars = 20 µmTie2Cre-DsRed validation in the bone marrow cells. Bone marrow smear preparation showed that Tie2Cre-DsRed marks a population of monocytes (A) and that these DsRed+ bone marrow cells were FSP1+ (B) but not αSMA+ (C). Scale bars = 20 µmMinimal renal fibrosis in wild-type mice. αSMA staining in wild-type mice showed that the majority of αSMA+ structures were arteries, afferent arterioles and renal pelvis smooth muscle. Scale bars = 20 µmLineage tracing revealed that a proportion of tubulointerstitial myofibroblast in the Crim1KST264/KST264 kidneys were Tie2-derived. (A) In the TZWT kidneys, minimal fibrosis was observed within the interstitium. (B, B′, C, D) In the TZCrim1 kidneys, however, there was a marked expansion of the DsRed+ cells within the interstitium and a proportion of the myofibroblasts were of Tie2 origin. Scale bars = 20 µmGlomerular cystic fibrosis in the Crim1KST264/KST264 kidneys was not primarily derived from the Tie2 population. (A) In the TZWT glomeruli, minimal fibrosis and restricted αSMA staining was observed in the arterioles. (B, C) The fibrosis occurring around the TZCrim1 glomeruli, however, was not of Tie2 origin. Scale bars = (A, C) 20 µm; (B) 0.1 µmSupplementary materials and methods istex:7B576A945D25F76000A309A54E3C5AD948E52903 ark:/67375/WNG-RGGCKWBZ-2 Joint senior authors. No conflicts of interest were declared. ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
| ISSN: | 0022-3417 1096-9896 |
| DOI: | 10.1002/path.4155 |