Fate tracing reveals the pericyte and not epithelial origin of myofibroblasts in kidney fibrosis

Abstract

Understanding the origin of myofibroblasts in kidney is of great interest because these cells are responsible for scar formation in fibrotic kidney disease. Recent studies suggest epithelial cells are an important source of myofibroblasts through a process described as the epithelial-to-mesenchymal transition; however, confirmatory studies in vivo are lacking. To quantitatively assess the contribution of renal epithelial cells to myofibroblasts, we used Cre/Lox techniques to genetically label and fate map renal epithelia in models of kidney fibrosis. Genetically labeled primary proximal epithelial cells cultured in vitro from these mice readily induce markers of myofibroblasts after transforming growth factor beta(1) treatment. However, using either red fluorescent protein or beta-galactosidase as fate markers, we found no evidence that epithelial cells migrate outside of the tubular basement membrane and differentiate into interstitial myofibroblasts in vivo. Thus, although renal epithelial cells can acquire mesenchymal markers in vitro, they do not directly contribute to interstitial myofibroblast cells in vivo. Lineage analysis shows that during nephrogenesis, FoxD1-positive((+)) mesenchymal cells give rise to adult CD73(+), platelet derived growth factor receptor beta(+), smooth muscle actin-negative interstitial pericytes, and these FoxD1-derivative interstitial cells expand and differentiate into smooth muscle actin(+) myofibroblasts during fibrosis, accounting for a large majority of myofibroblasts. These data indicate that therapeutic strategies directly targeting pericyte differentiation in vivo may productively impact fibrotic kidney disease.

Figure 1

Control and day 14 unilateral ureteral obstruction kidneys from Six2-GC; R26R and HoxB7-Cre; R26R mice show no evidence for LacZ genetically labeled epithelial cells migrating into the interstitium. A: Bigenic Six2-GC; Z/Red mice activate GFPCre expression in renal progenitor cells present in metanephric mesenchyme as they differentiate into epithelial cells. Differentiation results in removal of the LoxP-LacZ-STOP-LoxP sequence in epithelial cells, leading to permanent, heritable expression of RFP in epithelial cells. HoxB7-Cre; Z/Red mice activate Cre in the ureteric bud during embryonic development. Cre activation removes the LoxP-LacZ-STOP-LoxP sequence in collecting duct cells, leading to permanent, heritable expression of RFP in collecting duct epithelial cells. For bigenic Six2-GC; R26R mice, or HoxB7-Cre; R26R mice, removal of the LoxP-STOP-LoxP sequence leads to permanent, heritable expression of the LacZ gene in epithelial cells. B: Representative light microscopy images of X-gal stain indicative of LacZ expression in control or day 14 UUO fibrotic kidneys of R26R mice, HoxB7-Cre; R26R mice Six2-GC; R26R mice and R26-LacZ positive control mice. Low power view (left panels) of whole kidneys stained identically with X gal solution indicates the high level of recombination and the high signal-to-noise ratio generated by LacZ staining. High power view (center panels) of control kidneys shows specificity of labeling and lack of interstitial cells deriving from epithelial cells. Note all interstitial and vascular cells as well as epithelial cells label with blue stain in the R26-LacZ kidneys (inset shows blue-stained glomerulus). High power view (right panels) of day 14 UUO kidneys. Note no staining in the interstitium of HoxB7Cre; R26R or Six2-GC; R26R mice but homogenous blue stain throughout the interstitium of positive control R26-LacZ kidneys.

Figure 2

Day 10 unilateral ureteral obstruction kidneys from Six2-GC; Z/Red and HoxB7-Cre; Z/Red mice show no evidence for RFP+ genetically labeled epithelial cells, becoming interstitial cells expressing αSMA+. Representative confocal images of native RFP epifluorescence (left panels) or fluorescein isothiocyanate-conjugated antibody detection of αSMA (green, center panels) of day 10 UUO fibrotic kidneys of Z/Red mice, HoxB7-Cre; Z/Red mice Six2-GC; Z/Red mice and NLS-Cre-Z/Red mice. Composite images with nuclei in blue are seen (right panels). Note that all interstitial cells expressing αSMA co-express RFP in the NLS-Cre-Z/Red positive control mice, but no αSMA+ cells co-express RFP in the other mice. Scale bar = 25 μm.

Figure 3

Day 10 unilateral ureteral obstruction kidneys from Six2-GC; Z/Red and HoxB7-Cre; Z/Red mice show no evidence for RFP+ genetically labeled epithelial cells, becoming interstitial cells or acquiring the interstitial cell marker S100A4. Representative confocal images of native RFP epifluorescence (left panels) or fluorescein isothiocyanate-conjugated antibody detection of S100A4 (green, center panels) of day 10 UUO fibrotic kidneys of Z/Red mice, HoxB7-Cre; Z/Red mice Six2-GC; Z/Red mice and NLS-Cre-Z/Red mice. Composite images with nuclei in blue are seen (right panels). Note that all interstitial cells expressing S100A4 co-express RFP in the NLS-Cre-Z/Red positive control mice, but no S100A4+ cells co-express RFP in the other mice. Scale bar = 25 μm.

Figure 4

Cultured tubule cells from kidneys of Six2-GC; Z/Red mice, co-express αSMA and S100A4 cultured in the presence of transforming growth factor-β1. A: Day 10 cultured Six2-GC; Z/Red tubule cells fluoresce red from RFP expression, but do not label for αSMA. Scale bar = 50 μm. B: Day 10 cultured Six2-GC; Z/Red tubule cells grown in the presence of TGFβ₁ for 4 days fluoresce red with RFP and co-label for αSMA (green). C: Day 10 cultured Z/Red tubule cells grown in the presence of TGFβ₁ for 4 days show no evidence of RFP expression but co-label with αSMA (green). D: Day 10 cultured Six2-GC; Z/Red tubule cells grown in the presence of TGF1 for 4 days fluoresce red with RFP show no evidence of non-specific binding with irrelevant IgG control primary antibodies. E: Split panel images (right shows green channel only) Day 10 cultured Six2-GC; Z/Red tubule cells fluoresce red with RFP and do not label for S100A4. F: Split panel images showing Day 10 cultured Six2-GC; Z/Red tubule cells grown in the presence of TGFβ₁ for 4 days fluoresce red with RFP and co-label for S100A4 (green) in the cytoplasm and nucleus. G: Day 10 cultured Six2-GC; Z/Red tubule cells fluoresce red with RFP and label for E-cadherin (green). H: Day 10 cultured Six2-GC; Z/Red tubule cells grown in the presence of TGFβ₁ for 4 days also fluoresce red with RFP and co-label for E cadherin (green), however some cells lose E-cadherin expression.

Figure 5

Characterization of FoxD1 metanephric mesenchyme-derived cells in the adult kidney of FoxD1-GC; R26R mice. A: Bigenic FoxD1-GC; R26R mice activate GFPCre expression in renal progenitor cells that are fated to remain as stromal cells, removing the LoxP-STOP-LoxP sequence in stromal cells, leading to permanent, heritable expression of LacZ in stromal cells. B: Low power and higher power views of adult kidney cortex showing LacZ⁺, blue stained arteriolar smooth muscle and many perivascular cells surrounding peritubular capillaries. C: Images (split panel light and fluorescence microscopy) of normal adult kidney showing co-expression of LacZ with Pdgfrβ or CD73. D: Graph showing the proportion of Prgfrβ+, or CD73+ labeled interstitial cells co-expressing LacZ and the proportion of LacZ interstitial cells co-expressing either Pdgfrβ or CD73. E: Low-magnification image of FoxD1-GC; R26R mouse kidney following 14 days of ureteral obstruction. Note extensive expansion of interstitial LacZ⁺, blue-stained cells, but no staining of the epithelial compartment. F: Low magnification split light and fluorescence image showing co-expression of LacZ+ blue cells with αSMA (red) after UUO day 14. G: Graph showing quantification of the proportion of αSMA cells that co-express LacZ and the proportion of LacZ cells that co-express αSMA after kidney UUO day 14. H: RT-PCR for endogenous Foxd1 transcripts (40 cycles) compared with Gapdh (32 cycles), in day 10 pup kidney, normal adult kidney and adult kidney at times after UUO injury. Note absence of Foxd1 in normal adult kidney but gene expression following injury (n = 5/group). Scale bar = 100 μm.

Figure 6

Characterization of FoxD1 metanephric mesenchyme derived interstitial pericytes in the adult kidney of FoxD1-GCE; R26R mice. A: Bigenic FoxD1-GCE; R26R mice express GFP CreERT2 fusion protein (green) under control of the endogenous FoxD1 regulatory sequences in metanephric mesenchyme fated to differentiate into stromal cells. In the absence of tamoxifen, the fusion protein is excluded from the nucleus and does not mediate recombination between loxP sites. During a tamoxifen (Tam) pulse, GFPCreERT2 translocates to the nucleus activating fate marker expression, leading to permanent, heritable expression of LacZ in a cohort kidney stromal cells. B: Light and fluorescence photomicrographs showing LacZ expressing cells in the normal kidney of FoxD1-GCE; R26R mice treated with Tam on E10.5 only. Note positive cells (arrows) are exclusively in the interstitium and express the markers CD73, Pdgfrβ, but do not express αSMA or CD31. C: FoxD1 fated mesenchyme cells also become kidney arteriolar VSMCs and glomerular mesangial cells (left and center panels). Scale bar = 50 μm. D: Graph showing the proportion of Prgfrβ+, CD73+, αSma+, CD31+ labeled interstitial cells co-expressing LacZ and the proportion of LacZ interstitial cells co-expressing Pdgfrβ, CD73, αSma, CD31 in normal kidneys of FoxD1-GCE; Rs26R mice.

Figure 7

FoxD1-derived kidney pericytes/perivascular fibroblasts are myofibroblast progenitors following unilateral ureteral obstruction. A: Low magnification images of LacZ stained normal (left) and day 10 UUO (center) kidney FoxD1-GCE; R26R kidney treated with tamoxifen in utero on e10.5. Note marked expansion of interstitial blue stain following UUO. In day 10 UUO FoxD1-GCE; R26R kidney treated with vehicle (oil) on e10.5 there is no interstitial blue staining. (B, C) Representative split image light and fluorescence micrographs of day 10 UUO kidney structures from FoxD1-GCE; R26R mice treated on e10.5 with tamoxifen, showing anti-αSMA-Cy3 immunolabeling and anti-Pdgfrβ-Cy3 labeling. Note many αSMA+ or Pdgfrβ+ interstitial cells co-express LacZ (*denotes arteriole). D: Graph of area of LacZ staining in control (day 0) kidneys, or kidneys day 4 and day 10 following UUO from FoxD1-GCE; R26R mice treated on e10.5 with tamoxifen (n = 6/group). Note that the total area of LacZ+ cells increases markedly in the interstitium in response to UUO, but that without tamoxifen injection on e10.5 there are no LacZ cells in control of diseased kidneys. E: Graph showing the percentage of LacZ+ cells that co-express αSMAon day 10 of UUO disease and the percentage of αSMA+ cells that co-express LacZ. F: Graph showing the percentage of LacZ+ cells that co-express Pdgfrβ with time of disease and the proportion of Pdgfrβ+ cells that co-express LacZ with time of disease (n = 6/group). Scale bar = 50 μm. *P < 0.05.