Progenitor Renin Lineage Cells are not involved in the regeneration of glomerular endothelial cells during experimental renal thrombotic microangiopathy

Figures

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  Fig 1. Reversible glomerular EC damage in mRen-rtTAm2/LC1/LacZ mice during thrombotic microangiopathy-like injury model. Mice were analyzed at baseline (day 0), day 1, and day 7 following disease induction. A. Quantification of intraglomerular fibrin thrombi (pinkish) as revealed by Acid Fuchsin Orange G (AFOG) staining of kidney slices. Data are presented as mean ± SEM, n = 5/5/10 for day 0 /day 1 /day 7, respectively. ***p<0.001, ns- not significant by one-way ANOVA; B. Representative AFOG staining images in the course of the EC model. Scale bars correspond to 25 μm; C. Quantification of glomerular EC depletion and injury on kidney slices by erythroblast transformation-specific related gene (ERG) and platelet endothelial cell adhesion molecule-1 (CD31) staining, respectively. Data are presented as mean ± SEM, n = 5/5/10 for day 0 /day 1 /day 7, respectively. *p<0.05, **p<0.01, ns- not significant by one-way ANOVA; D. Representative ERG/CD31 co-staining images in the course of the EC model. 4′,6-diamidino-2-phenylindole (DAPI) was used as a nuclear marker. Scale bars correspond to 25 μm.

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  Fig 2. RLCs invade the glomerulus during EC model. A. Intraglomerular RLCs tagged by β-gal, but negative for renin appear in the regenerative phase of the EC model (day 7). Representative confocal microscopy images for day 0 and day 7 of β-gal/renin co-stained kidney slices. 4′,6-diamidino-2-phenylindole (DAPI) was used as a nuclear marker. The channels for green (β-gal) and red (renin) fluorescent signals in the dashed square on day 7 are separately shown in the small right panels. Scale bars correspond to 25 μm; B. Representative 3D reconstruction of glomeruli and β-gal labelled RLCs (blue) on day 0 and day 7 of the EC model. The mesangial cell marker α8-integrin (red) was used to visualize the glomeruli. Scale bars correspond to 20 μm; C. Quantification of glomeruli with tufts containing β-gal expressing RLCs in the regenerative phase of the EC model (day 7). Data are presented as mean ± SEM, n = 5/10 for day 0 (baseline) /day 7, respectively. n.d.—not detectable; D. The intraglomerular RLCs observed during the EC model are not of hematopoietic origin. Representative confocal microscopy images for day 0 and day 7 of β-gal/CD45 (hematopoietic marker) co-stained kidney slices.…

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  Fig 3. RLCs selectively differentiate to intraglomerular mesangial cells during EC model. A. Representative confocal microscopy images for day 0 and day 7 of β-gal and the EC markers ERG (upper panels) or CD31 (lower panels) co-stained kidney slices; B. Representative confocal microscopy images for day 0 and day 7 of β-gal and the mesangial cell markers PDGFβR (upper panels) or α8-integrin (lower panels) co-stained kidney slices; C. Representative confocal microscopy images for day 0 and day 7 of β-gal/WT1 (podocyte marker) co-stained kidney slices. 4′,6-diamidino-2-phenylindole (DAPI) was used as a nuclear marker throughout. The channels for green (β-gal) and red (corresponding cell marker) fluorescent signals in the dashed squares on day 7 are separately shown in the small right panels. Scale bars correspond to 25 μm.

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  Fig 4. Mesangial cells and not podocytes display considerable transient damage in the course of the EC model. Mice were analyzed at baseline (day 0), day 1, and day 7 following disease induction. A. Quantification of glomerular mesangial injury by α8-integrin (mesangial cell marker) staining of kidney slices. Data are presented as mean ± SEM, n = 5/5/10 for day 0 /day 1 /day 7, respectively. **p<0.01, ***p<0.001 by one-way ANOVA; B. Representative α8-integrin staining images in the course of the EC model. Scale bars correspond to 25 μm; C. Quantification of podocyte depletion and injury on kidney slices by WT1 and nephrin staining, respectively. Data are presented as mean ± SEM, n = 5/5/10 for day 0 /day 1 /day 7, respectively. *p<0.05, **p<0.01, ns- not significant by one-way ANOVA; D. Representative WT1/nephrin co-staining images in the course of the EC model. 4′,6-diamidino-2-phenylindole (DAPI) was used as a nuclear marker. Scale bars correspond to 25 μm.