Dysfunction of fibroblasts of extrarenal origin underlies renal fibrosis and renal anemia in mice

Abstract

In chronic kidney disease, fibroblast dysfunction causes renal fibrosis and renal anemia. Renal fibrosis is mediated by the accumulation of myofibroblasts, whereas renal anemia is mediated by the reduced production of fibroblast-derived erythropoietin, a hormone that stimulates erythropoiesis. Despite their importance in chronic kidney disease, the origin and regulatory mechanism of fibroblasts remain unclear. Here, we have demonstrated that the majority of erythropoietin-producing fibroblasts in the healthy kidney originate from myelin protein zero-Cre (P0-Cre) lineage-labeled extrarenal cells, which enter the embryonic kidney at E13.5. In the diseased kidney, P0-Cre lineage-labeled fibroblasts, but not fibroblasts derived from injured tubular epithelial cells through epithelial-mesenchymal transition, transdifferentiated into myofibroblasts and predominantly contributed to fibrosis, with concomitant loss of erythropoietin production. We further demonstrated that attenuated erythropoietin production in transdifferentiated myofibroblasts was restored by the administration of neuroprotective agents, such as dexamethasone and neurotrophins. Moreover, the in vivo administration of tamoxifen, a selective estrogen receptor modulator, restored attenuated erythropoietin production as well as fibrosis in a mouse model of kidney fibrosis. These findings reveal the pathophysiological roles of P0-Cre lineage-labeled fibroblasts in the kidney and clarify the link between renal fibrosis and renal anemia.

Figure 1. Most of the fibroblasts in the kidney arise from P0-Cre–expressing precursors.

(A) Schematic drawing of the kidney, showing the localization of P0-Cre fate-mapped cells in the cortex and outer medulla. (B and C) ECFP⁺ cells were detected in the interstitium of the kidneys of P0-Cre/R26ECFP mice. A higher-magnification view is shown in C. (D–F) LacZ staining of P0-Cre/R26R kidney. (D and E) Note the similar distribution of LacZ⁺ cells in P0-Cre/R26R kidneys with ECFP⁺ cells in P0-Cre/R26ECFP mice. A higher-magnification view is shown in E. (F) No LacZ⁺ cells were observed in the kidneys of R26R mice without the P0-Cre allele. (G–I) Double immunostaining of ECFP⁺ cells in the P0-Cre/R26ECFP kidney. (G) Most, if not all, of the ECFP⁺ cells were negative for PECAM. (H and I) ECFP⁺ cells (H) in the kidney were positive for PDGFR-β, the fibroblast marker, whereas ECFP⁺ cells (I) in the cortex were also positive for CD73/5′NT, the marker for cortical fibroblasts. (J) Graph illustrating the proportion of PDGFR-β⁺ interstitial cells in the cortex and outer medulla coexpressing ECFP (% ECFP/PDGFR-β cells) and the proportion of ECFP⁺ interstitial cells coexpressing PDGFR-β (% PDGFR-β/ECFP cells). (K) In neonatal kidneys of P0-Cre/floxed-EGFP mice most of the EGFP⁺ cells were also positive for p75, a neural crest marker. Scale bars: 10 μm (B–I); 100 μm (K).

Figure 2. P0-Cre lineage-labeled cells in the developing kidney.

(A–C) ECFP⁺ cells were first observed in the kidneys of P0-Cre/R26ECFP mice at E13.5. ECFP⁺ cells were predominantly located along the outer capsule and the ureter (arrows) of the kidney. (D–F) The number of ECFP⁺ cells in the kidneys of P0-Cre/R26ECFP mice increased at E16.5, and (F) the cells began to express PDGFR-β. The boxed region in E is shown at higher magnification in F. (G and H) ECFP⁺ cells populating the cortex appeared to surround the territory of Six2⁺ renal progenitor cells. (I) ECFP⁺ cells in the kidney were negative for class III β-tubulin. K, kidney. Scale bars: 100 μm (A, B, D, E, and I); 10 μm (C and F–H).

Figure 3. P0-Cre lineage-labeled fibroblasts produce EPO.

(A–C) Stellate-shaped GFP⁺ cells with projections were detected (A) in the interstitium of the kidneys of Epo-GFP mice and were positive for (B) CD73 and (C) PDGFR-β. (D and E) Analysis of the kidney of P0-Cre/R26R/Epo-GFP mice revealed that GFP⁺ cells were also positive for LacZ. (F) Analysis of the kidney of P0-Cre/R26tdRFP/Epo-GFP mice revealed that most GFP⁺ cells were also positive for tdRFP. Scale bars: 10 μm. (G) Sorting EGFP⁺ cells (P5) out of EGFP^– cells (P4) from the kidneys of adult P0-Cre/floxed-EGFP mice. RT-PCR analysis of these populations revealed high expression of Epo and p75 in EGFP⁺ cells but not in EGFP^– cells. Cells were stained either with or without anti-GFP antibody. (H) Sorting p75⁺ cells (P5) out of p75^– cells (P4) from the kidneys of P0-Cre/R26ECFP mice at 2 weeks of age. Cells were stained either with or without anti-p75 antibody. Quantitative PCR analysis demonstrated that the expression of p75, Epo, and ECFP was higher in p75⁺ cells. Expression of p75 was normalized to that of Gapdh and expressed relative to that in p75^– cells.

Figure 4. P0-Cre lineage-labeled fibroblasts transdifferentiate into myofibroblasts and contribute to fibrosis.

(A and B) Five days after obstruction of the ureter, (B) interstitial fibrosis was prominent in the operated kidney (A) compared with that in the control kidney. (C and D) The number of ECFP⁺ cells was markedly increased in the operated kidneys of P0-Cre/R26ECFP mice compared with that in the control kidney. (E and F) FACS analysis also confirmed the increase in the number of EGFP⁺ cells in the operated kidneys of P0-Cre/floxed-EGFP mice. Cells were stained with anti-GFP antibody. (G) ECFP/Ki67 double-positive cells were abundant in the operated kidneys of P0-Cre/R26ECFP mice. (H and I) Most ECFP⁺ cells were also positive for (H) PDGFR-β and (I) α-SMA. (J) Graph illustrating the proportion of PDGFR-β⁺ or α-SMA⁺ interstitial cells coexpressing ECFP (% labeled/ECFP cells) and the proportion of interstitial ECFP⁺ cells coexpressing either PDGFR-β or α-SMA (% ECFP/labeled cells). (K and L) The number of ECFP⁺ cells was also markedly increased in the kidneys of P0-Cre/R26ECFP mice after (K) folic acid nephrotoxicity and (L) severe ischemic reperfusion injury. Most ECFP⁺ cells were also positive for α-SMA. Scale bars: 10 μm.

Figure 5. EPO-producing fibroblasts also transform into myofibroblasts, hindering EPO production.

(A) In situ hybridization demonstrated that Epo mRNA detected in the control kidney was almost undetectable in the operated kidneys at day 14 of UUO. Hybridization with a sense probe did not elicit any signals in the control kidney. AS, antisense; S, sense. (B) Expression of Epo mRNA in the operated kidney decreased significantly, as early as 12 hours after ligation of the ureter (n = 5/group, except for the group sacrificed at day 5 [d5], in which n = 4). NT, nontreated. (C) The suppression of Epo mRNA in the operated kidneys was reversed after the induction of anemia. Each dot represents renal expression of Epo in an individual mouse (n = 5 in UUO group; n = 9 in UUO plus anemia group). (D) Undetectable GFP⁺ cells in the operated kidneys of Epo-GFP mice became detectable after the induction of anemia and colocalized with α-SMA. (E) The expression of Epo in cultured myofibroblasts was augmented by the administration of low-dose dexamethasone, neurotrophins, and HGF. Vehicle used was 0.05% ethanol for the control of dexamethasone. Data are representative of results of 5 independent experiments. (F) Expression of Epo mRNA in UUO kidneys was significantly restored, whereas the expression of Col1a1 and fibronectin (Fn1) mRNA was decreased by tamoxifen treatment (n = 5). Vehicle used was 100 μl of 10% ethanol in sunflower oil. Expression of Epo, Col1a1, and Fn1 was normalized to that of Gapdh and expressed relative to that in (B and E) the nontreated group or (F) vehicle-treated group. Scale bars: 10 μm.

Figure 6. Hypothetical model for 2 different characteristics of P0-Cre lineage-labeled fibroblasts in the kidney, with a possible therapeutic implication for treatment of renal anemia.

P0-Cre lineage-labeled fibroblasts in the kidney produce EPO, which possesses hematopoietic and tissue-protective functions in healthy kidneys. In contrast, they transdifferentiate into scar-producing myofibroblasts and lose EPO-producing activity after kidney injury, leading to renal fibrosis and renal anemia. We further demonstrated that the administration of various neuroprotective and renoprotective reagents restores the EPO-producing ability in fibrotic kidneys.