Renal Interstitial Platelet-Derived Growth Factor Receptor- β Cells Support Proximal Tubular Regeneration

Abstract

Background: The kidney is considered to be a structurally stable organ with limited baseline cellular turnover. Nevertheless, single cells must be constantly replaced to conserve the functional integrity of the organ. PDGF chain B (PDGF-BB) signaling through fibroblast PDGF receptor-β (PDGFRβ) contributes to interstitial-epithelial cell communication and facilitates regenerative functions in several organs. However, the potential role of interstitial cells in renal tubular regeneration has not been examined.

Methods: In mice with fluorescent protein expression in renal tubular cells and PDGFRβ-positive interstitial cells, we ablated single tubular cells by high laser exposure. We then used serial intravital multiphoton microscopy with subsequent three-dimensional reconstruction and ex vivo histology to evaluate the cellular and molecular processes involved in tubular regeneration.

Results: Single-tubular cell ablation caused the migration and division of dedifferentiated tubular epithelial cells that preceded tubular regeneration. Moreover, tubular cell ablation caused immediate calcium responses in adjacent PDGFRβ-positive interstitial cells and the rapid migration thereof toward the injury. These PDGFRβ-positive cells enclosed the injured epithelium before the onset of tubular cell dedifferentiation, and the later withdrawal of these PDGFRβ-positive cells correlated with signs of tubular cell redifferentiation. Intraperitoneal administration of trapidil to block PDGFRβ impeded PDGFRβ-positive cell migration to the tubular injury site and compromised the recovery of tubular function.

Conclusions: Ablated tubular cells are exclusively replaced by resident tubular cell proliferation in a process dependent on PDGFRβ-mediated communication between the renal interstitium and the tubular system.

Keywords: proliferation; renal proximal tubule cell; renal stem cell; tubular-interstitial crosstalk.

Figure 1.

Resident tubular cells undergo distinct morphologic changes and migrate in response to laser-induced tubular cell ablation. To investigate if and how resident proximal tubular cells respond to local tubular injury, we used partially induced Pax8-iCre × Confetti mice with single-tubular cell expression of CFP (blue), GFP (green), YFP (yellow), or RFP (red). Serial two-photon imaging (A and C) before and (B and D) 2 days after tubular cell ablation revealed characteristic morphologic changes of the adjacent tubular epithelium. Single Confetti-positive tubular cells turned from a cubical to a narrower and flask-like shape (arrowheads), which was accompanied by a distinct cell migration toward the injury site (arrows; average migration velocity: 1.87±0.14 μm/h; n=8).

Figure 2.

Resident tubular cells proliferate in response to laser-induced tubular cell ablation. To investigate whether tubular cells proliferate in response to laser-induced cell ablation, we performed (A and B) cell lineage tracing by serial imaging of Pax8-iCre × Confetti mice followed by (C and D) three-dimensional reconstruction. Injection of Hoechst 33342 allowed for the counting of tubular cell nuclei associated with monochromatic areas within the injured tubular epithelium. Note the different distribution pattern of the RFP-positive area (B and D, arrows) 7 days after tubular cell ablation (A and C) compared with baseline conditions. (B and D, arrows) Although originally located to more remote areas, 7 days after tubular cell ablation, monochromatic cell clusters were found within the injury site, suggesting tubular cell migration and clonal proliferation. (B) Quantification of tubular cell nuclei associated with the RFP-positive area before and 7 days after tubular cell ablation further suggested proliferation of resident tubular epithelial cells. Scale bars, 20 μm. (E and F) To further assess injury-induced tubular cell proliferation, we performed intravital serial imaging of CyclinB1-GFP reporter kidneys. In this genetic mouse model, replicating cells express GFP during the S, G2, and M phases of the cell cycle. (E) Five minutes after tubular cell ablation, there were no GFP-positive cells within or around the injury site (asterisk). (F) In contrast, 2 days after tubular cell ablation, a strong epithelial GFP signal (arrows) was detected in the affected tubular epithelium, indicating proliferation of resident tubular cells after local cell ablation. Furthermore, shed autofluorescent epithelial material of the injured tubular segment was found in the tubular lumen.

Figure 3.

Functional proximal tubule recovery involves epithelial cell dedifferentiation. Serial intravital imaging of a low dose–induced Pax8-iCre × Confetti kidney (A) 5 minutes and (B) 2 days after single-cell ablation revealed characteristic morphologic changes of migrating tubular cells. Compared with baseline conditions, injury-adjacent RFP- and YFP-positive tubular cells (B, arrows) turned from a cubical into a flat cell shape and migrated toward the injury site (A, asterisk). (C) Seven days after tubular cell ablation, the injury-responding tubular cells were localized at the site of injury and had regained a normal cubical cell morphology (arrows). Scale bar, 20 μm. Serial intravital imaging of Pax8-iCre × Confetti kidneys (D) before and (E) 2 days after tubular cell ablation again showed the (E, arrowheads) flattening and (E, arrows) migration of injury-adjacent RFP-positive tubular cells (2 days after tubular cell ablation). (F) Two days after cell ablation, the same RFP-positive cell was reidentified ex vivo in the fixed kidney tissue (arrowheads), and (G) it showed the expression of the dedifferentiation marker CD44 (cyan and arrows). Scale bar, 20 μm. (I) Seven days after tubular cell ablation, reidentification and CD44 staining (cyan) of the regenerated tubule (arrows) on fixed kidney tissue labeled only few interstitial cells (arrowhead) and revealed no epithelial expression of CD44. Different distribution pattern of Confetti-positive cells (H) at baseline and (I) 7 days after cell ablation further suggested migration. Note the clonal cell division of a single YFP-positive cell (I, arrows) at the site of injury (nuclei are labeled with Hoechst). Scale bar, 20 μm. (J and L) Two and (K and M) 7 days after laser-induced cell ablation, tubular epithelium integrity and function were assessed using serial intravital 2-PM. (J and K) The tubular epithelium was visualized by collecting autofluorescence (gray). (L and M) Subsequently, FITC albumin was injected in vivo (green), and proximal tubular albumin reuptake was determined. (J) Two days after cell ablation (asterisk), cell material was shed from the adjacent tubular epithelium (arrows). (L) Compromised tubular epithelial integrity was associated with strong functional impairment as indicated by the severe reduction of in vivo–injected FITC albumin uptake (green) within and adjacent to the injury site (arrowheads). Seven days after injury, the affected tubular epithelium had (K) morphologically (arrows) and (M) functionally (green FITC albumin uptake at the injury site) recovered. (N) Quantification of proximal tubular albumin uptake capacity at the injury site 1, 2, and 7 days after laser-induced cell ablation. Values are expressed as percentage of control proximal tubular albumin uptake. **P<0.01; ***P<0.001.

Figure 4.

Interstitial PDGFRβ cells are motile and recruited to the tubular injury site. To investigate the response of interstitial PDGFRβ cells to laser-induced tubular cell ablation, we performed in vivo serial imaging (insets) followed by three-dimensional reconstruction of PDGFRβ-iCre × mTmG kidneys at (A) baseline, (B) 1 day, (C) 2 days, and (D) 7 days after laser-induced injury. Interstitial PDGFRβ cells (cyan) were quickly recruited toward the injury site (purple) and enclosed the affected tubular epithelium within 1–2 days after laser-induced tubular cell ablation. (D) Seven days after injury, PDGFRβ cells had redispersed into the renal interstitium. Scale bars, 30 μm.

Figure 5.

PDGFRβ cell activation occurs predominantly in close proximity of the tubular injury site. Fully induced PDGFRβ-iCre × GCaMP5 mice, which coexpress tdTomato (red) and the fluorescent [Ca²⁺] indicator protein GCaMP5G (cyan) in PDGFRβ-positive cells (arrows), were investigated (A) before and (B) after laser-induced tubular cell ablation. Proximal tubules were visualized by collecting autofluorescence. (B) Note the strong increase of GCaMP5G fluorescence intensity (arrows and inset) immediately after tubular injury, whereas tdTomato fluorescent intensity remained unchanged (inset), indicating a strong increase of PDGFRβ [Ca²⁺] values. Scale bar, 20 μm. (C) Changes of PDGFRβ [Ca²⁺] levels in response to tubular cell ablation were expressed as the GCaMP5G-to-tdTomato ratio in percentage of baseline values. ***P<0.001 for 100-μm radius versus 200- and 375-μm radii. (D–F) To further investigate PDGFRβ [Ca²⁺] levels during interstitial cell recruitment, we performed serial intravital 2-PM imaging followed by three-dimensional reconstruction of PDGFRβ-iCre × GCaMP5 mice. (D) Three-dimensional reconstruction 5 minutes after tubular cell ablation revealed that initial PDGFRβ cell activation (high [Ca²⁺] levels; cyan and arrows) occurred predominantly in PDGFRβ cells with cell-cell contact to the injured tubular epithelium. (E) Within the first 24 hours, these early injury-responding PDGFRβ cells migrated toward the injury site (arrows). (E and F) Twenty-four to 48 hours after injury, additional PDGFRβ cells had been activated and recruited toward the tubular injury site (cyan cells). Scale bars, 30 μm. (G) [Ca²⁺] levels of PDGFRβ cells in close proximity of the injury site given as the GCaMP5G-to-tdTomato ratio in percentage of baseline values (n=25 each). There were no changes in PDGFRβ cell [Ca²⁺] in control areas. ***P<0.001 for ablated versus control tubules.

Figure 6.

PDGFRβ cell recruitment is accompanied by resident proximal tubular cell dedifferentiation. Serial 2-PM intravital imaging of PDGFRβ-iCre × mTmG mice (A) before and (B) 2 days after laser-induced tubular cell ablation confirmed recruitment of interstitial PDGFRβ cells (cyan) to the injury site (arrowheads). (C) In vivo injection of FITC-conjugated albumin (cyan) revealed functional impairment of the PDGFRβ cell–enclosed tubular epithelium (arrows in the inset). Scale bars, 20 μm. (D) To determine if reduced tubular albumin uptake capacity was a sign of resident tubular epithelial cell dedifferentiation, the same cortical area was reidentified ex vivo on fixed tissue and stained for the dedifferentiation marker CD44. Ex vivo histology showed epithelial CD44 expression (yellow and arrows) within the PDGFRβ cell–enclosed tubular epithelium, which colocalized with the malfunctional area observed in vivo (arrows). PDGFRβ cells are shown in cyan, and tdTomato is shown in red. Scale bars, 20 μm.

Figure 7.

PDGFRβ cell withdrawal from the site of injury is accompanied by resident proximal tubular cell redifferentiation. To investigate the timeline of proximal tubular injury and repair in relation to PDGFRβ cell dynamics, we performed serial intravital 2-PM imaging of PDGFRβ-iCre × mTmG mice before (not shown) and (A and B) 2 and (C and D) 7 days after laser-induced tubular cell ablation. (A and B) Two days after injury, PDGFRβ cell recruitment (arrowheads) to the injury site (asterisks) was accompanied by reduced in vivo FITC albumin uptake (arrows). (C and D) However, 7 days after injury, PDGFRβ cells had completely withdrawn from the injury site (arrowheads), and proximal tubular function had recovered (arrows). (E) Ex vivo histology of the regenerated tubule on fixed tissue revealed little to no epithelial CD44 staining (yellow) within the regenerated tubular segment (arrows in the inset), suggesting resident tubular cell redifferentiation. Few interstitial CD44-positive cells (arrowheads) confirmed the successful staining. Scale bars, 20 μm.

Figure 8.

PDGFRβ-blockade compromises interstitial cell recruitment and recovery of proximal tubular function. (A) Effects of trapidil, a competitive PDGFRβ inhibitor, on PDGFRβ cell [Ca²⁺] level 3, 6, 12, 24, and 48 hours after laser-induced proximal tubular injury (n=25 each). **P<0.01 for trapidil-treated versus untreated tubules. (B) Trapidil treatment reduced PDGFRβ cell migration velocity compared with untreated experiments. Proximal tubule of a trapidil-treated animal 7 days after laser-induced tubular cell ablation (C) before and (D) after in vivo FITC albumin injection (cyan). (C) Unlike in untreated animals, PDGFRβ cells (cyan) had not redispersed after 7 days into the renal interstitium, but they still enclosed the injury site (arrows). In addition, the tubular epithelium contained large vacuoles at the injury site (arrowheads). (D) Seven days after injury, FITC albumin injection revealed scattered areas of functional (cyan FITC albumin uptake; arrowheads) and malfunctional albumin uptake (reduced or absent cyan FITC albumin uptake; arrows). Scale bar, 20 μm. (E) Trapidil treatment reduced the recovery of proximal tubular albumin reuptake capacity 7 days after injury.