Early activation of fibroblasts is required for kidney repair and regeneration after injury

Abstract

Acute kidney injury (AKI) is a devastating condition with high morbidity and mortality. AKI is characterized by tubular injury, inflammation, and vascular impairment. However, the role of interstitial fibroblasts in the pathogenesis of AKI is largely unknown. Here, we show that fibroblasts were activated, as defined by vimentin expression, at 1 h after AKI triggered by ischemia-reperfusion injury (IRI). They rapidly entered the cell cycle with Ki-67-positive staining, which started at 1 h and peaked at 12 h after IRI, whereas tubular cell proliferation peaked at 3 d. The trigger for such an early activation of fibroblasts was identified as sonic hedgehog (Shh), which was rapidly induced in renal tubules and could target interstitial fibroblasts. Tubule-specific knockout of Shh in mice inhibited fibroblast activation and aggravated kidney injury and functional decline after IRI. Likewise, pharmacologic inhibition of Shh signaling with cyclopamine also hindered fibroblast activation and exacerbated kidney damage. These studies uncover that tubule-derived Shh triggers the early activation of fibroblasts, which is required for kidney repair and regeneration. Our findings for the first time illustrate a previously unrecognized importance of interstitial fibroblasts in conferring renal protection in AKI.-Zhou, D., Fu, H., Liu, S., Zhang, L., Xiao, L., Bastacky, S. I., Liu, Y. Early activation of fibroblasts is required for kidney repair and regeneration after injury.

Keywords: Wnt–β-catenin; acute kidney injury; injury repair; sonic hedgehog.

Figure 1

Activation of interstitial fibroblasts is an early event after AKI. A, B) Immunohistochemical staining showed the vimentin‐positive fibroblasts at different time points (1, 4, and 12 h, and 1, 3 and 7 d) after IRI (A). Enlarged boxes are presented (B) that indicate vimentin expression at 1 h after IRI compared with the sham‐treated controls. Black arrows indicate vimentin‐positive cells. C) Bar graph showed the numbers of vimentin‐positive cells in each high‐power field at different time points after IRI. **P < 0.01 vs. sham‐treated (n =3). D) Representative micrographs show renal a‐SMA‐positive cell population at different time points after IRI. Kidney sections were immunostained with antibody against a‐SMA. Scale bar, 50 μm. E) Bar presentation of the numbers of the a‐SMA‐positive myofibroblasts at different time points after IRI. Shown are the cell numbers per high‐power field. **P < 0.01 vs. sham‐treated (n = 3). F) Serum creatinine levels at different time points after IRI. *P < 0.05 vs. sham‐treated controls (n = 4). G) Tubular injury index (percentage of injured tubules) at different time points after IRI. Tubular injury was defined by loss of brush borders, cell flatting, and cell death or detachment. *P < 0.05 vs. sham‐treated controls (n = 4). H) Realtime qPCR showed renal expression of NGAL mRNA at different time points after IRI. *P < 0.05 vs. sham‐treated controls (n = 3‐4). Ctrl, control; SCr, serum creatinine.

Figure 2

Fibroblast proliferation precedes tubular repair and regeneration after AKI. A, B) Immunohistochemical staining for Ki‐67 showed that fibroblast proliferation is an early event preceding tubular regeneration. A) Representative micrographs of renal Ki‐67 staining at different time points after IRI are shown. Scale bar, 50 μm. B) Enlarged boxes are presented. Arrows indicate Ki‐67‐positive cells in renal interstitium. C) Double immunostaining showed renal Ki‐67 and cell type‐specific markers, such as vimentin, α‐SMA, F4/80, and CD31, at 12 h after IRI. White arrows indicate Ki‐67‐positive cells. D) Coimmunostaining with antibodies against Ki‐67 and laminin demonstrated tissue compartment‐specific cell proliferation at different time points after IRI. Small arrowheads indicate interstitial cells, whereas large arrowheads indicate tubular epithelial cells. E) Graphic presentation showed different dynamics of fibroblast and tubular cell proliferation after AKI. Ctrl, control.

Figure 3

Tubule‐derived Shh triggers fibroblast activation in AKI. A, B) Shh was induced specifically in renal tubules as early as 1 h after IRI. A) Kidney sections at different time points after IRI were immunostained with anti‐Shh antibody. Scale bar, 50 μm. B) Boxed areas were enlarged and presented. Arrow indicates positive staining. C‐F) Shh signaling was rapidly activated in the kidney after AKI. Renal expression of Shh (C), Gli1 (D), Patch1 (E), and Hhip (F) mRNA at different time points after IRI was assessed by real‐time qPCR. **P < 0.01, *P < 0.05 vs. sham‐treated controls (n =3‐4). G) Shh targets interstitial fibroblasts in AKI. The Gli1‐LacZ reporter mice were subjected to IRI. At 1 d after IRI, kidney sections were subjected to X‐gal staining. Arrows indicate X‐gal‐positive interstitial cells in renal interstitium. Scale bar, 50 μm. Ctrl, control.

Figure 4

Shh induction precedes renal Wnt‐β‐catenin activation after AKI. A, B) Representative micrographs (A) show immunostaining of β‐catenin at different time points after IRI, as indicated. Scale bar, 50 μm. Enlarged boxes (B) show β‐catenin expression at 1 d after IRI. Arrow indicates positive staining. C, D) Western blot analyses demonstrated renal Shh and β‐catenin expression at different time points after IRI. Representative Western blot (C) and relative abundance of Shh and β‐catenin (D) are shown. E, F) Real‐time qPCR showed renal expression of Wnt‐β‐catenin targeted genes in the kidney after IRI. Relative abundance of renal Snail1 (E) and fibronectin (FN) (F) mRNA are presented. *P < 0.05 vs. sham‐treated controls (n =3‐4). G) Shh induces the expression of Wnt ligands in cultured interstitial fibroblasts. NRK‐49F cells were incubated with different doses of human recombinant Shh protein for 24 h, as indicated. The mRNA expression of various Wnt ligands was assessed by RT‐PCR. H‐J) Shh induces HGF expression in renal interstitial fibroblasts. NRK‐49F cells were incubated with Shh (50 ng/ml) for various periods of time as indicated. HGF mRNA (H) and protein levels (I, J) are shown. *P < 0.05 vs. sham‐treated controls (n = 3). Ctrl, control.

Figure 5

Blockade of Shh signaling inhibits fibroblast activation and aggravates AKI after IRI. A) Experiment design. CPN was administrated in mice 2 d prior to IRI, and mice were euthanized 1 d after IRI. B) Body weight changes after CPN administration. C) CPN abolished renal Gli1 mRNA induction at 1 d after IRI. *P < 0.05 vs. sham‐treated controls, +P < 0.05 vs. vehicle (n = 5, sham‐treated n = 3). D) Blockade of Shh signaling by CPN aggravated kidney dysfunction after IRI. Serum creatinine levels were assessed at 1 d after IRI in different groups as indicated. **P < 0.01, ⁺ P < 0.05 (n = 9, sham‐treated n =3). E) Real‐time qPCR revealed an increased NGAL mRNA expression in mice injected with CPN at 1 d after IRI compared with vehicle controls. *P< 0.05 vs. sham‐treated controls, +P < 0.05 vs. vehicles (n = 5, sham‐treated n = 3). F) Inhibition of Shh signaling by CPN worsened histologic injury after AKI. Representative micrographs show kidney histologic changes at 1 d after IRI. G) Bar presentation show the percentage of injured tubules in different groups as indicated. **P < 0.01, +P < 0.05 vs. control (n = 3‐5). H) Representative micrographs show that inhibition of Shh signaling blocked fibroblast activation and proliferation. Kidney sections were stained with antibodies against vimentin and Ki‐67. Arrows indicate positive staining. Scale bar, 50 μm. I, J) Western blot analysis showed renal PDGFR‐β levels in different groups at 1 d after IRI. Representative Western blot (I) and quantitative data (J) are shown. **P < 0.01 vs. sham‐treated controls, +P < 0.05 vs. vehicle (n =3‐5). K) Representative RT‐PCR showed renal mRNA levels of Wnt ligands in different groups as indicated at 1 d after IRI. PAS, periodic acid Schiff; SCr, serum creatinine; Veh, vehicle.

Figure 6

Tubule‐specific ablation of Shh inhibits fibroblast activation and aggravates AKI. A) Schematic diagram of the strategy used to generate tubule‐specific, Shh‐conditional knockout mice. Uninjured kidney histology was presented by periodic acid Schiff staining. B) Real‐time qPCR revealed a dramatic reduction of Shh mRNA in Ksp‐Shh^‐/‐ kidney at 1 d after IRI compared with the controls. *P < 0.05 (n =5‐6). C) Representative micrographs show Shh protein expression at 1 d after IRI in the control and Ksp‐Shh^‐/‐ mice. Arrows indicate positive staining. Scale bar, 50 μm. D‐F) Tubule‐specific ablation of Shh inhibited its downstream target genes after IRI. Renal expression of Gli1 (D), Patch1 (E), and tenascin C (TNC) (F) mRNA at 1 d after IRI was analyzed by real‐time qPCR in the control and Ksp‐Shh^‐/‐ mice. *P < 0.05 (n =5‐6). G) Changes in serum creatinine at 1 d after IRI in the control and Ksp‐Shh^‐/‐ mice. *P < 0.05 (n =7‐9). H) Histologic changes of the control and Ksp‐Shh^‐/‐ kidneys at 1 d after IRI. Blue asterisks indicate injured tubules. I) Quantitative data show the percentage of injured tubules in the control and Ksp‐Shh^‐/‐ mice. *P < 0.05 (n = 7). J‐M) Western blot analyses showed renal levels of PDGFR‐β, a‐SMA, and β‐catenin proteins at 1 d after IRI. Representative Western blot (J) and quantitative data on PDGFR‐β (K), a‐SMA (L), and β‐catenin (M) are presented. *P < 0.05 (n = 5). N) Representative micrographs show renal expression of β‐catenin protein in the control and Ksp‐Shh^‐/‐ mice. Arrows indicate positive staining. O, P) Real‐time qPCR revealed substantial reductions of fibroblast‐specific protein 1 (FSP‐1), vimentin, and plasminogen activator inhibitor 1 mRNA levels in Ksp‐Shh^‐/‐ kidney at 1 d after IRI compared with the controls. *P < 0.05 (n =5‐6). R) Representative micrographs show renal Ki‐67‐positive cells at 1 d after IRI in the control and Ksp‐Shh^‐/‐ mice. Arrows indicate positive staining. Scale bar, 50 μm. S) Quantitative data show the Ki‐67‐positive cells at 1 d after IRI in the control and Ksp‐Shh^‐/‐ mice. *P < 0.05 (n = 3). Ctrl, control; SCr, serum creatinine.

Figure 7

Interstitial fibroblasts are activated in human kidney biopsies of patients with AKI. A) Serum Shh levels in patients with AKI (n = 9) compared with the healthy subjects (n = 9). *P < 0.05 (n = 9). B) Representative micrographs showed protein expression of Shh, vimentin (Vim), a‐SMA, and β‐catenin in human kidney biopsy specimens from patients with AKI. Nontumor kidney tissue from the patients who had renal cell carcinoma and underwent nephrectomy was used as normal controls. Arrows indicate positive staining. Scale bar, 50 μm. C) Diagram depicts the renoprotective role of early activation of fibroblasts in AKI. After injury, kidney tubular cells express and secrete Shh, which targets and activates interstitial fibroblasts, highlighted by vimentin expression, as early as 1 h after IRI. Activated fibroblasts then undergo proliferation (Ki‐67‐positive) peaking at 12 h and secrete Wnt ligands and activate β‐catenin in tubular cells. This leads tubular cell proliferation and tubular repair and regeneration peaking at 3 d after IRI. Pharmacologic inhibition or genetic ablation of Shh blunts fibroblast activation and proliferation and aggravates kidney injury after AKI. Ctrl, control.