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

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.


Mouse models of AKI
As previously described S1 , 8-to 10-week-old Gli1-Smo+/+ and Gli1-Smo-/-or Pdgfrβ-Smo+/+ and Pdgfrβ-Smo-/mice were intraperitoneally administrated tamoxifen (T5648, Sigma-Aldrich, St. Louis, MO) at 30 mg/kg for 5 consecutive days to induce target Smo gene ablation.After 1 week of washout, these mice were subjected to IRI or Cisplatin injection following an established protocol S1-3 .In brief, after mice were anesthetized, a midline abdominal incision was made, and bilateral renal pedicles were clipped for 30 minutes using microaneurysm clamps.The mouse body temperature was maintained between 36°C and 37.5°C using a temperature-controlled heating system during the ischemic period.
Mice were euthanized at 1 day after IRI, and serum and kidney tissues were collected for various analyses.The cisplatin-induced AKI model involved a single intraperitoneal injection of cisplatin at 30 mg/kg body weight, and then mice were sacrificed 3 days after injection.

Determination of Serum Creatinine
Serum was collected from mice at 1 day after IRI.Serum creatinine level was determined using a QuantiChrom creatinine assay kit, according to the protocols specified by the manufacturer (BioAssay Systems, Hayward, CA).The serum creatinine level was expressed as milligrams per 100 ml (dL).Of note, the baseline serum creatinine levels of Gli1-Smo+/+, Gli1-Smo-/-, Pdgfrβ-Smo, and Pdgfrβ-Smo-/-mice (n=6/group) were measured by high-performance liquid chromatography at the George O'Brien Kidney Center at Yale University.
Mice were placed in the specialized holder for 10 -15 minutes before the measurement to acclimate to their surroundings.Heating pads, supplied as part of the Visitech Systems BP2000 system, were preheated to 37°C.Before initial baseline measurements, all animals underwent one-week (twice/day: daytime and night) training.

Human Kidney Biopsy Specimens
Human kidney specimens were obtained from diagnostic kidney biopsies performed at the Presbyterian Hospital of the University of Pittsburgh Medical Center.Nontumor kidney tissue from patients with renal cell carcinoma who underwent nephrectomy was used as normal controls.All patients in the presented study signed the informed consent forms before they underwent kidney biopsy or nephrectomy.The Institutional Review Board approved all studies involving human kidney sections at the University of Pittsburgh and the University of Connecticut School of Medicine.

Histology and Immunohistochemical Staining
Paraffin-embedded mouse kidney sections (3 μm thickness) were prepared by a routine procedure.The sections were stained with periodic acid-Schiff staining reagents by standard protocol.
Immunohistochemical staining was performed according to the established protocol as described previously S6 .After incubation with primary antibodies at 4°C overnight, the slides were then stained with HRP-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA).
Non-immune normal IgG was used to replace primary antibodies as a negative control, and no staining was visible.Slides were viewed under an Olympus BX43 microscope equipped with a digital camera (Allentown, PA).The image quantification was independently performed by two experienced technicians.The detailed information of antibodies used is presented in Supplementary Table S5.

Immunofluorescence staining
Kidney cryosections were fixed with 3.7% paraformaldehyde for 15 min at room temperature.NRK-49F and NRK-52E cells cultured on coverslips were fixed with cold methanol:acetone (1:1) for 10 min at room temperature.After blocking with 10% donkey serum for 1 hour, the slides were immunostained with primary antibodies.These slides were then stained with Cy2-or Cy3-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA).Slides were viewed under an Olympus BX43 microscope equipped with a digital camera or an Olympus FluoView 1000 confocal microscope.The image quantification was independently performed by two experienced technicians.The detailed information of antibodies used is presented in Supplementary Table S5.

Detection of apoptotic cells
Apoptotic cell death was determined by using TUNEL staining with a DeadEnd Fluorometric Apoptosis Detection System (Promega, Madison, WI) or immunostaining with anti-cleaved caspase 3 (#9664S, Cell Signaling Technology, Danvers, MA), as described previously S7-9 .The cell number counting for the positive apoptotic cells was independently performed by two experienced technicians.

Western Blot Analysis
Kidney tissues were lysed with radioimmune precipitation assay (RIPA) buffer containing 1% NP-40, 0.1% SDS, 100 μg/ml PMSF, 1% protease inhibitor cocktail, and 1% phosphatase I and II inhibitor cocktail (Cell Signaling Technology, Danvers, MA) in PBS on ice.The supernatants were collected after centrifugation at 13,000×g at 4°C for 15 min.Protein expression was analyzed by western blot analysis as described previously S10 .The detailed information of antibodies used is presented in Supplementary Table S5.

Quantitative Real-Time Reverse Transcription PCR (qRT-PCR)
Total RNA isolation and qRT-PCR were carried out by procedures described previously S10 .Briefly, the first strand cDNA synthesis was carried out using a reverse transcription system kit according to the instructions of the manufacturer (Promega).qRT-PCR was performed on an ABI PRISM 7000 sequence detection system (Applied Biosystems, Foster City, CA).The mRNA levels of various genes were calculated after normalizing with β-actin.Primer sequences used for amplifications are presented in Supplementary Table S4.

LC-MS/MS analysis
The LC-MS/MS analysis was performed using an Ultimate 3000 nanoLC and Q Exactive mass spectrometer system (Thermo Scientific).Peptides were separated on in-house packed capillary columns (19 cm length × 75 μm ID; Reprosil C18, 3 μm) at a flow rate of 200 nl/min.The mobile phases were (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile.Gradient elution was performed as follows: 1 to 35% B in 180 min, 35 to 80% B in 10 min, stay at 80% for 5 min, and then back to 2% B in 5 min followed by 20 min equilibration.Spray voltage was 1700 V and the scan range was m/z 350-1800.The top 10 most intense precursor ions were selected for MS/MS using HCD at 30% energy with an automatic gain control value of 1e5, a maximum injection time of 120 ms, and detection at a mass resolution of 60,000 at m/z 200 in the orbitrap analyzer.MS/MS scans were acquired with an automatic gain control value of 5e4 and a maximum injection time of 120 ms.
Dynamic exclusion was set for 30 s with a 10 ppm gate with detection at a mass resolution of 15,000 at m/z 200 in the Orbitrap analyzer.

Protein identification and quantitation.
MaxQuant-Andromeda software suite (version 1.6.3.4) was used for protein identification and quantitation with most of the default parameters S14 .The mass spectra were searched against a mouse database (17,038 sequences; Reviewed only; version March 2019) downloaded from UniProt Knowledgebase (https://www.uniprot.org/).For global proteome analysis, the following parameters were applied: trypsin as enzyme with two missed cleavage sites; 10 ppm and 20 ppm mass tolerances for precursor and fragments, respectively; protein N-terminal acetylation and methionine oxidation as variable modifications; cysteine carbamidomethylation as a fixed modification; peptide length with at least 7 amino acids.False discovery rate (FDR) was set at 1% for both proteins and peptides.The MaxQuant output (proteinGroups.txt)was log2-transformed prior to downstream data analyses such as missing value imputation, Hierarchical clustering, Principal Component Analysis (PCA), t-tests, correlation, and volcano plots, which were performed in the Perseus environment using default parameters (version 1.6.2.3).For phosphoproteome analysis, phosphorylation at serine, threonine, and tyrosine was set as an additional variable modification during MaxQuant database search.The cutoff of phosphosite probability estimated by MaxQuant was required to be 0.75 or higher.The MaxQuant output file (Phospho (STY) Sites.txt) was utilized for further bioinformatics analyses and were performed in the Perseus environment as well.
Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed using DAVID Bioinformatics Resources 6.8.For protein network analysis, the StringApp was employed in the Cytoscape environment (version 3.8.0)S15 .The interaction score was set to 0.9, the highest confidence cutoff, to retrieve potential interactions.The 'Load Enrichment Data' option was enabled during this process to retrieve functional enrichment (minimum significance threshold FDR 0.05) for the STRING network, as we described previously S11 .

Enzyme-linked immunosorbent assay (ELISA)
The rat NID-1 (MBS2886242) Elisa kit was purchased from MyBioSource, Inc (San Diego, CA).This assay employs the quantitative sandwich enzyme immunoassay technique.Antibody specific for NID1 has been pre-coated onto a 96-well strip plate.We prepared 7 wells for standard, 1 well for blank and added 100 μl each of standard, blank, and sample dilutions into the appropriate wells.The plate was then incubated at 37°C for 2 hours.Liquid from each well was removed and 100μl of Detection Reagent A working solution was added to each well.The plate was incubated again at 37°C for 1 hour.
After washing, 100Μl of Detection Reagent B working solution was added to each well and the plate was incubated for 30 minutes at 37°C.Following a wash to remove any unbound reagent, 90 μl of substrate solution was added to the wells, and color developed in proportion to the amount of NID1 bound in the initial step.The color development was stopped, and the intensity of the color was measured immediately using a microplate reader set to 450 nm.

ZDOCK and RDOCK algorithms
The crystal structure of Smo was downloaded from RCSB Protein Data Bank (PDB code 4JKV), and the predicted structure of NID1 is downloaded at AlphaFold Protein Structure Database (https://www.alphafold.ebi.ac.uk/).The Dock Proteins (ZDOCK) and Refine Docked Proteins (RDOCK) protocols of Discovery Studio 2021 (DS2021, Accelrys, CA, USA) were conducted to the virtual protein-protein interactions.The ZDOCK protocol provides rigid body docking of two protein structures using the ZDOCK algorithm S17 .RDOCK optimizes and scores a set of docked protein poses from ZDOCK via a CHARMm-based procedure S18 .RDock energy, an energy scoring function, was employed to score these binding protein complexes.For RDock results, the RDock energy (E_RDock) is divided into two parts as the equation.E_sol represents the desolvation energy based on the Atomic Contact Energy.E_elec2 denotes the Electrostatic energy after second minimization with ionic residues in charged state, and β is a scaling factor, set as default value 0.9 S19 .The lower the RDOCK energy of the complex, the more likely it is in the optimal conformation.Equation: E_RDock = E_sol + β*E_elec2

Fibroblast decellularized ECM scaffold preparation
Serum-starved NRK-49F cells under CoCl2 induced-hypoxia stress were treated with or without CPN or transfected with Dicer-substrate smo-siRNA for 24 h.Cells were then treated with EGTA (#3889; Sigma, St Louis, MO) (0.5 mM, PH=7.4) in calcium-free PBS, followed by shaking at 4°C for 1h.The treatment was repeated 3-4 times until all cells were lifted from their underlying matrix.The fibroblast decellularized ECM scaffold was rinsed with PBS and then stored at 4°C for further experiments, as previously described S20 .Some ECM scaffolds were collected by scraping with a rubber policeman in loading buffer for western blot assay to detect NID-1 content.All ex vivo experiments were repeated three times at least.

Molecular docking study
Amino acid sequences for human Nidogen-1 (NID1) and Integrin β1 (ITGB1) were retrieved from the UniProt databank with ID P14543 and P09055, respectively.Currently, the NID1 and ITGB1 protein crystal structures were not resolved in the RCSB PDB protein crystal database.However, the crystal structures with high resolutions could be used as homology modeling templates to construct 3D for NID1 (1gi4/90.91%)and ITGB1 (7nxd/92.23%),respectively.
The following three steps (blind docking and precise docking) were implanted to explore possible binding interface between NID1 and ITGB1.Rosetta software was used for docking simulations S21 .
The docking protocol began with a rigid-body blind docking in which all heavy atoms of protein NID1 and ITGB1 were strictly position restrained.In this step, we randomly placed the proteins roughly facing each other, and at least 2,000 docking conformations were obtained for further analysis.The rigid docking conformations were clustered by using root-mean-square deviation (RMSD) values, and finally the clustered position with highest ranking ratio was selected as a potential binding spot.
Binding spots far away from the others were deleted manually.Using this approach, we were able to achieve an acceptable-quality prediction for the NID1 "binding pocket" of ITGB1.Rosetta software is most accurate when docking locally.In precise docking, the aptamer was randomly placed (within ~10 Å) within the binding pockets, and then the input structure of the aptamer was perturbed by 3 Å translation and 8° rotation before the start of every individual simulation.During precise docking, the side chains of the protein residues at the binding pocket and the complete NID1 were allowed to move.
Finally, a maximum number of 100 conformers were considered, and the conformation with the lowest binding energy in two binding spots were selected for final molecular dynamics simulations.
To check the NID1-ITGB1 complex stability, the best docking result in each binding spots were employed for molecular dynamics (MD) simulations.It was carried out by AMBER software (version 16), using AMBER ff99SB force field for complex S22 .Hydrogen atoms were added to the initial NID1-ITGB1 complex model using the leap module, setting ionizable residues as their default protonation states at a neutral PH value.The complexes were both solvated in a cubic periodic box of explicit TIP3P water model that extended a minimum 10 Å distance from the box surface to any atom of the solute.The particle mesh Ewald (PME) method for simulation of periodic boundaries was used to estimate the long-range electrostatic interactions with a cutoff of 10.0 Å.All bond lengths were constrained using the SHAKE algorithm and integration time step was set to 2fs using the Varlet leapfrog algorithm.To eliminate possible bumps between the solute and the solvent, the entire systems was minimized in two steps.Firstly, the complex was restrained with a harmonic potential of the form k (Δx)2 with a force constant k =100 kcal/mol-1Å-2.The water molecules and counter ions were optimized using the steepest descent method of 2,500 steps, followed by the conjugate gradient method for 2,500 steps.Secondly, the entire system was optimized by using the first step method without any constraint.These two minimization steps were followed by annealing simulation with a weak restraint (k=100kcal/mol-1Å-2) for the complex and the entire system was heated gradually in the NVT ensemble from 0 to 298K over 500ps.After the heating phase, a 10ns MD simulation was performed under 1atm.The constant temperature was selected at 298K with the NPT ensemble.Constant temperature was maintained using the Langevin thermostat with a collision frequency of 2ps-1.The constant pressure was maintained employing an isotropic position scaling algorithm with a relaxation time of 2ps.RMSD values were tested to monitor the conformation fluctuations of the NID1-ITGB1 complex.Based on the final 10ns MDs trajectory, 3000 snapshots were extracted from the last 3ns trajectory for the final average structure of the complex.

Statistics
All data were expressed as mean ± SEM if not specified otherwise in the legends.Statistical analysis of the data was performed using GraphPad Prism 9 (GraphPad Software, San Diego, CA).Comparison between two groups was made using a two-tailed Student's t-test or the Rank Sum Test if data failed a normality test.Statistical significance for multiple groups was assessed by one-way ANOVA, followed by the Student-Newman-Keuls test.Results are presented in dot plots, with dots denoting individual values.Exact P values are presented for all dot plots.P < 0.05 was considered statistically significant.
timers are set for 12-hour light/12-hour dark.Animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh School of Medicine and the University of Connecticut School of Medicine.