. 2024 May 7;134(13):e165836.

doi: 10.1172/JCI165836.

Fibroblast expression of transmembrane protein smoothened governs microenvironment characteristics after acute kidney injury

Yuan Gui¹, Haiyan Fu², Zachary Palanza¹, Jianling Tao³, Yi-Han Lin⁴, Wenjian Min⁵, Yi Qiao⁶, Christopher Bonin⁷, Geneva Hargis⁷, Yuanyuan Wang¹, Peng Yang⁵, Donald L Kreutzer⁶, Yanlin Wang¹, Yansheng Liu^{8

9}, Yanbao Yu¹⁰, Youhua Liu², Dong Zhou¹

Affiliations

¹ Division of Nephrology, Department of Medicine, University of Connecticut School of Medicine, Farmington, Connecticut, USA.
² Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
³ Division of Nephrology, Department of Medicine, Stanford University School of Medicine, Stanford, California, USA.
⁴ National Center for Advancing Translational Sciences, Rockville, Maryland, USA.
⁵ State Key Laboratory of Natural Medicines and Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing, China.
⁶ Department of Surgery and.
⁷ University of Connecticut, School of Medicine, Farmington, Connecticut, USA.
⁸ Yale Cancer Biology Institute, Yale University, West Haven, Connecticut, USA.
⁹ Department of Pharmacology, School of Medicine, Yale University, New Haven, Connecticut, USA.
¹⁰ Department of Chemistry & Biochemistry, University of Delaware, Newark, Delaware, USA.

PMID: 38713523
PMCID: PMC11213467
DOI: 10.1172/JCI165836

Fibroblast expression of transmembrane protein smoothened governs microenvironment characteristics after acute kidney injury

Yuan Gui et al. J Clin Invest. 2024.

. 2024 May 7;134(13):e165836.

doi: 10.1172/JCI165836.

Authors

Affiliations

¹ Division of Nephrology, Department of Medicine, University of Connecticut School of Medicine, Farmington, Connecticut, USA.
² Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
³ Division of Nephrology, Department of Medicine, Stanford University School of Medicine, Stanford, California, USA.
⁴ National Center for Advancing Translational Sciences, Rockville, Maryland, USA.
⁵ State Key Laboratory of Natural Medicines and Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing, China.
⁶ Department of Surgery and.
⁷ University of Connecticut, School of Medicine, Farmington, Connecticut, USA.
⁸ Yale Cancer Biology Institute, Yale University, West Haven, Connecticut, USA.
⁹ Department of Pharmacology, School of Medicine, Yale University, New Haven, Connecticut, USA.
¹⁰ Department of Chemistry & Biochemistry, University of Delaware, Newark, Delaware, USA.

PMID: 38713523
PMCID: PMC11213467
DOI: 10.1172/JCI165836

Abstract

The smoothened (Smo) receptor facilitates hedgehog signaling between kidney fibroblasts and tubules during acute kidney injury (AKI). Tubule-derived hedgehog is protective in AKI, but the role of fibroblast-selective Smo is unclear. Here, we report that Smo-specific ablation in fibroblasts reduced tubular cell apoptosis and inflammation, enhanced perivascular mesenchymal cell activities, and preserved kidney function after AKI. Global proteomics of these kidneys identified extracellular matrix proteins, and nidogen-1 glycoprotein in particular, as key response markers to AKI. Intriguingly, Smo was bound to nidogen-1 in cells, suggesting that loss of Smo could affect nidogen-1 accessibility. Phosphoproteomics revealed that the 'AKI protector' Wnt signaling pathway was activated in these kidneys. Mechanistically, nidogen-1 interacted with integrin β1 to induce Wnt in tubules to mitigate AKI. Altogether, our results support that fibroblast-selective Smo dictates AKI fate through cell-matrix interactions, including nidogen-1, and offers a robust resource and path to further dissect AKI pathogenesis.

Keywords: Extracellular matrix; Nephrology; Proteomics.

PubMed Disclaimer

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

**Figure 1. Fibroblast-specific ablation of Smo mitigates ischemic AKI.**
(A) Generation of Smo fibroblast-specific deletion mice. (B) Quantitative real-time PCR (qPCR) analysis (n = 4) and Western blot assay showing Smo levels in primary fibroblasts isolated from *Gli1-Smo^+/+* and *Gli1-Smo^–/–* or *Pdgfr*-β*-Smo^+/+* and *Pdgfr*-β*-Smo^–/–* kidneys. (C) The mouse baseline body weight (BW), kidney-to-body weight (KW/BW) ratios, serum creatinine (Scr), and (D) blood pressure levels (n = 6). At 1 day after IRI, (E) Scr levels in *Gli1-Smo^+/+* and *Gli1-Smo^–/–* mice (n = 16-17). (F) Periodic Acid–Schiff (PAS) staining showing kidney morphological changes in *Gli1-Smo^+/+* and *Gli1-Smo^–/–* mice. Blue asterisks indicate injured tubules. Representative micrographs of TUNEL or IHC staining against CD45 and CD3 in *Gli1-Smo^+/+* and *Gli1-Smo^–/–* kidneys. Scale bar: 25 μm. (G) Western blots assay of NGAL, FasL, and Bad in *Gli1-Smo^+/+* and *Gli1-Smo^–/–* kidneys (Sham, n = 3; IRI, n = 5). (H) qPCR analysis showing *Tnf-*α, *Mcp1*, and *Rantes* mRNA levels in *Gli1-Smo^+/+* and *Gli1-Smo^–/–* kidneys (Sham, n = 3; IRI, n = 6). (I) Scr levels in *Pdgfr*-β*-Smo^+/+* and *Pdgfr*-β*-Smo^–/–* mice (n = 15-16). (J) Western blots showing NGAL, FasL, and Bad levels in *Pdgfr*-β*-Smo^+/+* and *Pdgfr*-β*-Smo^–/–* kidneys. (K) qPCR analysis showing *Tnf-*α, *Mcp1*, and *Rantes* mRNA levels in *Pdgfr*-β*-Smo^+/+* and *Pdgfr*-β*-Smo^–/–* kidneys (Sham, n = 3; IRI, n = 6). (L) Representative micrographs of PAS, TUNEL, and IHC staining against CD45 in *Pdgfr*-β*-Smo^+/+* and *Pdgfr*-β*-Smo^–/–* kidneys. Scale bar: 25 μm. Arrows indicate positive cells. DAPI is a nuclear counterstain. For all Western blot panels, numbers indicate individual animals in a given group. †P < 0.05 versus sham control, *P < 0.05 versus *Gli1-Smo^+/+* or *Pdgfr*-β*-Smo^+/+* IRI mice. Dots indicate individual animals in a given group. Graphs are presented as means ± SEM. Differences among groups were analyzed using unpaired t tests or 1-way ANOVA followed by the Student-Newman-Keuls test.

**Figure 2. Fibroblast-specific ablation of Smo promotes perivascular mesenchymal cell activation and proliferation after AKI.**
At 1 day after IRI, (A) qPCR analyses of *Fsp1*, *vimentin*, and α*Sma* mRNA in *Gli1-Smo^+/+* and *Gli1-Smo^–/–* kidneys (Sham, n = 3; IRI, n = 7-12). (B) qPCR analyses of *Fsp1*, *vimentin*, and α*Sma* mRNA in *Pdgfr*-β*-Smo^+/+* and *Pdgfr*-β*-Smo^–/–* kidneys (Sham, n = 3; IRI, n = 6). (C) Western blot assay of Pdgfr-β, vimentin, αSMA, and PCNA proteins in *Gli1-Smo^+/+* and *Gli1-Smo^–/–* kidneys. (D) Western blots assay of vimentin, αSMA, and PCNA proteins in *Pdgfr*-β*-Smo^+/+* and *Pdgfr*-β*-Smo^–/–* kidneys. (E) Representative micrographs of Pdgfr-β, vimentin, and Ki67 expression in *Gli1-Smo^+/+* and *Gli1-Smo^–/–* kidneys. Arrows indicate positive cells. Scale bar: 25 μm. (F) Representative micrographs of vimentin and PCNA expression in *Pdgfr*-β*-Smo^+/+* and *Pdgfr*-β*-Smo^–/–* kidneys. Scale bar: 25 μm. Arrows indicate positive cells. DAPI is a nuclear counterstain. For all Western blot panels, numbers indicate individual animals in a given group. †P < 0.05 versus sham control, *P < 0.05 versus *Gli1-Smo^+/+* or *Pdgfr*-β*-Smo^+/+* IRI mice. Dots indicate individual animals in a given group. Graphs are presented as means ± SEM. Differences among groups were analyzed using 1-way ANOVA, followed by the Student-Newman-Keuls test.

**Figure 3. Global proteomics identifies NID1 as a prominent matrix protein in Gli1⁺ fibroblast-specific Smo deletion kidneys after AKI.**
(A) Experimental workflow of the global proteomic analysis. 6 mice in each group were used for mass spectrometry. (B) Principal component analysis of global proteomes from *Gli1-Smo^+/+* and *Gli1-Smo^–/–* kidneys 1 day after ischemic AKI. (C) Heatmap of ANOVA-significant proteins. Label-free quantitation (LFQ) intensity of represented proteins were z-scored and plotted according to the color bar. 2 clusters of proteins with different patterns of abundance profiles are highlighted in the heatmap. (D) GO cellular compartment terms in each cluster of proteins are plotted with their names and significance. ECM proteins are boxed to indicate the protein group with the largest difference in upregulated proteins. (E) Volcano plot shows the differential proteins between *Gli1-Smo^+/+* and *Gli1-Smo^–/–* kidneys. Up and down regulated proteins (FC, fold-change) are colored in red and blue, respectively. Yellow dots indicate ECM proteins. (F) Heatmap of differentially expressed ECM proteins. (G and H) Western blots of NID1 protein in *Gli1-Smo^+/+* and *Gli1-Smo^–/–* or in *Pdgfr*-β*-Smo^+/+* and *Pdgfr*-β*-Smo^–/–* kidneys 1 day after IRI. Numbers indicate individual animals in a given group. (I) Single nucleus RNA-Seq showing NID1 mainly expressed by fibroblasts (Fib) and pericytes (Per) 12 hours after IRI. (Data were extracted from the online database provided by Benjamin Humphrey’s laboratory, http://humphreyslab.com/SingleCell/displaycharts.php) (J) IHC staining showing NID1 protein expression in *Gli1-Smo^+/+* and *Gli1-Smo^–/–* or in *Pdgfr*-β*-Smo^+/+* and *Pdgfr*-β*-Smo^–/–* kidneys 1 day after IRI. Arrows indicate positive staining. Scale bar: 25 μm. (K) Immunoprecipitation revealing that Smo binds to NID1. NRK-49F cells under CoCl₂ stress were immunoprecipitated with Smo or NID1 antibody, followed by immunoblotting with antibody against NID1 (left blot) or Smo (right blot). Differences among groups were analyzed using unpaired t tests.

**Figure 4. Phosphoproteomics reveals Wnt pathway activation in fibroblast-specific Smo-deletion kidneys after AKI.**
(A) Workflow of phosphoproteomics. (B) Principal component analysis of phosphoproteins from *Gli1-Smo^+/+* and *Gli1-Smo^–/–* kidneys 1 day after ischemic AKI. (C) Volcano plot of pairwise comparisons (fold-change, FC) between the kidney phosphoproteomes of *Gli1-Smo^+/+* and *Gli1-Smo^–/–* kidneys 1 day after ischemic AKI. (D) qPCR of *Wnt 1*, 2, 4, 5A, 5B, 7A, 7B, 9B, *10A*, *10B*, 11, and 16 mRNA in *Gli1-Smo^+/+* and *Gli1-Smo^–/–* kidneys 1 day after ischemic AKI. *P < 0.05 (n = 7–12). (E) Western blots of β-catenin, Wnt 1, and Wnt 5A/B proteins in *Gli1-Smo^+/+* and *Gli1-Smo^–/–* kidneys 1 day after ischemic AKI. Numbers indicate individual animals in a given group. (F) Western blots of β-catenin and Wnt 1 in *Pdgfr*-β*-Smo^+/+* and *Pdgfr*-β*-Smo^–/–* kidneys 1 day after ischemic AKI. Numbers indicate individual animals in a given group. (G) IHC staining showing β-catenin, Wnt1, Wnt 4, and Wnt5A/B in *Gli1-Smo^+/+* and *Gli1-Smo^–/–* or in *Pdgfr*-β*-Smo^+/+* and *Pdgfr*-β*-Smo^–/–* kidneys 1 day after ischemic AKI. Scale bar: 25 μm. (H) IHC staining showing Wnt1, Wnt 4, and Wnt5A/B expression in kidney biopsy specimens from patients with AKI. Arrows indicate positive staining. Scale bar: 25 μm. Graphs are presented as means ± SEM. Differences between groups were analyzed using unpaired t tests.

**Figure 5. Deleting Smo in fibroblasts affects integrins linked to tubular cells after AKI.**
(A) Western blot assay showing the expression of integrin receptors linked to tubular cells, including integrin β1, α3, α6, αV, α8, and β6, in *Gli1-Smo^+/+* and *Gli1-Smo^–/–* kidneys after ischemic AKI at 1 day. Numbers indicate individual animals in a given group. (B and C) Representative Western blot showing the expression of integrin β1 in *Gli1-Smo^+/+* and *Gli1-Smo^–/–* (B) or in *Pdgfr*-β*-Smo^+/+* and *Pdgfr*-β*-Smo^–/–* (C) kidneys at 1 day after ischemic AKI. Numbers indicate individual animals in a given group. Quantitative data are accordingly presented in D and E. Dots indicate individual animals in a given group (Sham, n = 3; IRI, n = 6). †P < 0.05 versus sham control, *P < 0.05 versus *Gli1-Smo^+/+* or *Pdgfr*-β*-Smo^+/+* IRI mice. (F) IHC staining showing integrin β1 in *Gli1-Smo^+/+* and *Gli1-Smo^–/–* or in *Pdgfr*-β*-Smo^+/+* and *Pdgfr*-β*-Smo^–/–* kidneys at 1 day after AKI. Scale bar: 25 μm. (G) IHC staining showing NID1 and integrin β1 expression in kidney biopsy specimens from patients with AKI. Arrows indicate positive staining. Scale bar: 25 μm. Graphs are presented as means ± SEM. Differences among groups were analyzed using 1-way ANOVA, followed by the Student-Newman-Keuls test.

**Figure 6. Specific deletion of Smo in fibroblasts mitigates AKI induced by cisplatin.**
(A) Schematic diagram. i.p., intraperitoneal. At 3 days after cisplatin (Cis) injection, (B) Serum creatinine (Scr) levels in *Gli1-Smo^+/+* and *Gli1-Smo^–/–* mice (n = 12). (C) Scr levels in *Pdgfr*-β*-Smo^+/+* and *Pdgfr*-β*-Smo^–/–* mice (n = 12). (D and E) Western blots assay showing NGAL and Bax protein levels in *Gli1-Smo^+/+* and *Gli1-Smo^–/–* (D) or in *Pdgfr*-β*-Smo^+/+* and *Pdgfr*-β*-Smo^–/–* (E) kidneys. (F) Periodic Acid–Schiff (PAS) staining showing kidney morphological changes in *Gli1-Smo^+/+* and *Gli1-Smo^–/–* mice. Blue asterisks indicate injured tubules. Representative micrographs of TUNEL assay or IHC staining against CD45 in *Gli1-Smo^+/+* and *Gli1-Smo^–/–* kidneys. Scale bar: 25 μm. White arrows indicate apoptotic cells adn black arrows indicate CD45⁺ cells. (G) Representative micrographs of PAS staining, TUNEL assay, and IHC staining against CD45 in *Pdgfr*-β*-Smo^+/+* and *Pdgfr*-β*-Smo^–/–* kidneys. Scale bar: 25 μm. Blue asterisks indicate injured tubules. White and black arrows, respectively, indicate apoptotic cells and CD45⁺ cells. (H and I) Western blots assay of NID1, Wnt1, and integrin β1 proteins in *Gli1-Smo^+/+* and *Gli1-Smo^–/–* (H) or in *Pdgfr*-β*-Smo^+/+* and *Pdgfr*-β*-Smo^–/–* (I) kidneys. Quantitative data are accordingly presented in J and K (Sham, n = 3; Cis, n = 5). †P < 0.05 versus sham control, *P < 0.05 versus *Gli1-Smo^+/+* or *Pdgfr*-β*-Smo^+/+* mice. Dots indicate individual animals in a given group. (L) IHC staining showing NID1, Wnt1, and integrin β1 (Intg β1) in *Gli1-Smo^+/+* and *Gli1-Smo^–/–* or in *Pdgfr*-β*-Smo^+/+* and *Pdgfr*-β*-Smo^–/–* kidneys. Scale bar: 25 μm. Arrows indicate positive cells. DAPI is a nuclear counterstain. For all Western blot panels, numbers indicate individual animals in a given group. Graphs are presented as means ± SEM. Differences among groups were analyzed using 1-way ANOVA, followed by the Student-Newman-Keuls test.

**Figure 7. NID1 promotes Wnt components expression and reduces tubular cell death in vitro.**
(A–D) Normal rat kidney fibroblasts (NRK-49F) were transfected with Dicer-substrate Smo siRNA (Smo Dsi) or incubated with Smo inhibitor cyclopamine (CPN, 2.5 μM), then subjected to hypoxic stress (CoCl₂, 400 μM) for 24 hours. Compared to control siRNA (NC Dsi) or vehicles, western blots demonstrating that knockdown (A) or inhibition (B) of Smo induced Pdgfr-β, vimentin, αSMA, and PCNA expression, and increased NID1 (C and D). (E) Immunofluorescence staining showing Smo inhibition induced NID1 in fibroblasts under hypoxic stress. Scale bar: 25 μm. Arrows indicate positive staining. (F) Enzyme-linked immunosorbent assay revealed NID1 concentration after incubation with CPN under hypoxic stress (n = 6). (G) Schematic diagram. (H and I) Western blots demonstrating β-catenin, Wnt1, Wnt2, and Wnt5A/B levels after knockdown (H) or inhibition (I) of Smo under hypoxic stress. (J and K) Western blots showing that NID1 recombinant protein (rNID1) elevated β-catenin, Wnt1, Wnt5A/B, and Wnt16 in cultured normal rat kidney proximal tubular cells at different dosages under basal conditions (J) and hypoxic stress (K). (L, M, and O) After stimulation with staurosporine (1 μM) for 3 h, western blots assay showing reduced cleaved-caspase 3 (CCP3) in tubular cells incubated with NID1-enriched CM collected from Smo-knockdown (L) or CPN-treated (M) fibroblasts or directly treated with rNID1 (O). (N and P) Immunofluorescence staining showed fewer CCP3+ cells after treated with NID1-enriched CM (N) or NID1 recombinant protein (P). Quantitative data are presented in Q (n = 3, 4 random images were selected per slide, each dot represents the score of the according image). †P < 0.05 versus control, *P < 0.05 versus vehicle after STS. Scale bar: 25 μm. Cells were costained with DAPI. Arrows indicate positive staining. Graphs are presented as means ± SEM. Differences among groups were analyzed using 1-way ANOVA, followed by the Student-Newman-Keuls test.

**Figure 8. NID1-enriched decellularized fibroblast matrix scaffold activates Wnt components and reduces tubular cell death ex vivo.**
(A) Experimental design. In step 1, normal rat kidney fibroblasts (NRK-49F) were transfected with Dicer-substrate Smo siRNA (Smo Dsi) or cultured with Smo inhibitor CPN to enrich for NID1. In step 2, scaffolds were isolated, and, in step 3, normal rat kidney proximal tubular cells (NRK-52E) were seeded on top of scaffolds. (B) Western blots assay showing NID1 protein in decellularized fibroblast matrix scaffold. (C and D) Western blots assay showing β-catenin, Wnt2, and Wnt5A/B were activated in NRK-52E cells seeded on NID1-enriched matrix scaffold isolated from Smo-knockdown (C) or CPN-treated (D) fibroblasts under hypoxic stress. (E and F) Western blots assay showing NID1-enriched matrix scaffold isolated from Smo-knockdown (E) or CPN-treated (F) fibroblasts reduced cleaved caspase-3 (CCP3) in the seeded NRK-52E cells. (G–I) Western blot assay showing conditioned medium collected from Smo-knockdown fibroblasts (G) or decellularized fibroblast matrix scaffold isolated from Smo-knockdown fibroblasts (H) or NID1 recombinant protein (rNID1) increased integrin β1 in NRK-52E cells under hypoxic stress. (J and K) Under hypoxic stress, knockdown of integrin β1 using Dicer-substrate siRNA (integrin β1 Dsi) repressed β-catenin accumulation after incubation with rNID1 in NRK-52E cells (J) and induced CCP3 (K). (L) Molecular docking analysis showing the binding sites between NID1 and Integrin β1. (M) The strategy of designing a mutant form of NID1. (N and O) Compared with the active form of NID1 human recombinant protein (25 ng/mL), Western blot assay demonstrating that mutant NID1 (25 ng/mL) failed to induce β-catenin expression in NRK-52E cells under hypoxic stress (N) and increased CCP3 after staurosporine (1 μM) stimulation (O). (P) Our model illustrates that loss of fibroblast-selective Smo promotes NID1 to interact with tubular integrin β1 and subsequently activated the Wnt signaling pathway in tubules, forming a favorable microenvironment to protect against AKI.

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References

1. Ronco C, et al. Acute kidney injury. Lancet. 2019;394(10212):1949–1964. doi: 10.1016/S0140-6736(19)32563-2. - DOI - PubMed
1. Little M, Humphreys B. Regrow or repair: an update on potential regenerative therapies for the kidney. J Am Soc Nephrol. 2021;33(1):15–32. doi: 10.1681/ASN.2021081073. - DOI - PMC - PubMed
1. Kramann R, et al. Gli1+ pericyte loss induces capillary rarefaction and proximal tubular injury. J Am Soc Nephrol. 2017;28(3):776–784. doi: 10.1681/ASN.2016030297. - DOI - PMC - PubMed
1. Zhou D, et al. Early activation of fibroblasts is required for kidney repair and regeneration after injury. FASEB J. 2019;33(11):12576–12587. doi: 10.1096/fj.201900651RR. - DOI - PMC - PubMed
1. Chou YH, et al. Methylation in pericytes after acute injury promotes chronic kidney disease. J Clin Invest. 2020;130(9):4845–4857. doi: 10.1172/JCI135773. - DOI - PMC - PubMed

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Fibroblast expression of transmembrane protein smoothened governs microenvironment characteristics after acute kidney injury

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Fibroblast expression of transmembrane protein smoothened governs microenvironment characteristics after acute kidney injury

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