Home Nephro Research Connectivity mapping of glomerular proteins identifies dimethylaminoparthenolide as a new inhibitor of diabetic kidney disease

Connectivity mapping of glomerular proteins identifies dimethylaminoparthenolide as a new inhibitor of diabetic kidney disease

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Connectivity mapping of glomerular proteins identifies dimethylaminoparthenolide as a new inhibitor of diabetic kidney disease

Animals

Male C57BL/6-Ins2Akita/J (Ins2Akita) and C57BL/6J (WT) mice (Charles River) were housed with unrestricted access to water and were maintained on a 12-h light–dark cycle in a pathogen-free environment on standard mouse chow. All experiments were conducted in accordance with the Guide for the care and use of laboratory animals of the National Institute of Health, eighth edition and the French Institute of Health guidelines for the care and use of laboratory animals. The project was approved by the local (Inserm/UPS US006 CREFRE) and national ethics committees (ethics committee for animal experiment, CEEA122; Toulouse, France; approval 02867.01).

Treatments and urine collection

In a first series of experiments, 2 months old WT and Ins2Akita mice were treated with or without ACEi ramipril (10 mg/kg/d in drinking water) for 2 months. In a second series of experiments 2 months old WT and Ins2Akita mice were treated with or without ACEi ramipril as in the first series or with dimethylaminoparthenolide monofumarate (DMAPT, 10 mg/kg/d by gavage) for 2 months. In each series of experiments, urine was collected in metabolic cages for 12 h, few days before sacrifice.

Isolation of glomeruli

Glomeruli were isolated as previously described35 with minor modifications. Mice were anesthetized by ip injection of a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg) in phosphate buffered saline (PBS). A catheter was placed into the abdominal aorta after ligation of the cava vein, the upper abdominal aorta, the mesenteric and the celiac arteries. The lower part of the abdominal aorta was perfused with 40 ml of Dynabeads M-450 Tosylactivated (4.5 µm diameter, Dynal A.S., Oslo, Norway) at a concentration of 2 × 106 beads/ml followed by 15 ml of cold PBS. This procedures allows accumulation of beads in glomeruli36. Next, the left and right kidneys were collected and decapsulated. The animal was sacrificed by cervical dislocation and blood was collected by intra-cardiac puncture in heparinized tubes and plasma was prepared by centrifugation at 1500×g at 4 °C for 5 min and stored at − 20 °C for further use. One portion of the right kidney was snap-frozen in liquid nitrogen and stored at − 80 °C. Another portion of the right kidney cortex was fixed in Carnoy’s solution (ethanol/chloroform/glacial acetic acid: 60/ 30/10, v/v/v) for further histological analysis.

The left kidney was gently pressed manually through a 70 µm cell strainer using a flattened pestle followed by washing of the cell strainer with 20 ml of cold PBS. The filtrate was centrifuged at 200×g for 5 min at 4 °C, and the glomerular pellet was adjusted to 2 ml with PBS and transferred to an Eppendorf tube that was placed in a magnetic particle concentrator (Dynal A.S., Oslo, Norway) to concentrate the glomeruli into a pellet. The supernatant was discarded and the pellet was washed 5 × with 1 ml of PBS. The final pellet was resuspended in 100 µl PBS. This procedure allows the isolation of ~ 4,000 glomeruli per kidney.

Based on light microscopy survey, our glomerular suspensions were highly enriched for glomeruli (Figure S2A,B). Moreover, mRNA quantification showed that isolated glomeruli were enriched in glomerular-specific genes (nphs2, podxl and cldn5) while proximal (aqp1, slc22a13), distal tubules (wnk), and of loop of Henle (aqp1, slc12a3) specific genes were not, or poorly enriched compared to total kidney (Figure S2). Moreover, among the 2,422 proteins detected by LC-MS/MS, several glomerular-specific proteins were ranked within the first upper quartile of relative abundance (columns AR-AS, Table S1) including NphS1 (rank 196), Tjp1/ZO-1 (rank 221) Nphs2 (rank 339), Synpo (rank 388), Actn4 (rank 7), cd2ap (rank 543), while several tubular-specific proteins Aqp1 (rank 591), Umod (rank 1634) were ranked as less abundant proteins, or were not detected (slc22a13, Wnk). These observations support the relative high purity of our glomerular preparations.

Renal histology

Right kidney cortexes were fixed in Carnoy’s solution for 24 h, dehydrated in successive baths with increasing alcohol concentrations, embedded in paraffin and 2 µm sections were cut and mounted, and stained with either periodic acid-Schiff (PAS) or Masson trichrome. Sections were digitalized with a Nanozoomer 2.0 RS (Hamamatsu Photonics SARL) and glomerular surface was quantified with Morpho-expert software (version 1.00, Explora Nova). At least 50 glomeruli including superficial and juxtaglomerular cortical area, were examined for each animal. The extent of glomerular injury was expressed as the percentage of glomerular area fraction occupied by PAS positive matrix37.

Biochemical analysis

Urinary albumin concentration was measured by ELISA using the AlbuWell kit (WAK-Chemie Medical GmbH, Steinbach, Germany). Urinary creatinine concentration was measured by the colorimetric method of Jaffe according to the protocol Creatinine Assay Kit (Bio Assay Systems). Blood glucose levels were measured in caudal blood from fasted awake mice using a glucometer (Glucometer Elite XL; Bayer Healthcare, Elkhart, IN).

Quantitative proteomics of glomerular samples

Glomerular sample preparation for proteomics

Isolated glomeruli were homogenized in RIPA buffer under agitation for 3 min and centrifuged 15 min at 13,000×g to pellet the beads together with cell debris. The supernatants were collected and stored at − 80 °C at a protein concentration of 1–2 mg/ml before being processed for mass spectrometry (MS) analysis38. Protein samples were air-dried using a SpeedVac concentrator and reconstituted in 1 × final Laemmli buffer containing dithiothreitol (25 mM) and heated at 95 °C for 5 min. Cysteines were alkylated in chloroacetamide (75 mM) for 30 min at room temperature. Proteins (10 µg) were loaded onto a 12% acrylamide SDS-PAGE gel and concentrated in a single band visualized by Coomassie staining (Instant Blue—Expedeon). The gel band containing the whole sample was cut and washed in 50 mM ammonium bicarbonate:acetonitrile (1:1) for 15 min at 37 °C. Proteins were in-gel digested using 0.6 μg of modified sequencing-grade trypsin (Promega) in 50 mM ammonium bicarbonate overnight at 37 °C. Peptides were extracted from the gel by two incubations in 10% formic acid:acetonitrile (1:1) for 15 min at 37 °C. Extracted fractions were pooled with the initial digestion supernatant and dried under speed-vaccum. The resulting peptides were resuspended with 14 µL of 5% acetonitrile, 0.05% trifluoroacetic acid for nanoLC-MS/MS analysis.

NanoLC-MS/MS analysis

Peptides were analyzed by nanoLC-MS/MS using an UltiMate 3000 system (Dionex) coupled to an LTQ Orbitrap Velos ETD mass spectrometer (Thermo Fisher Scientific)38. Each sample (5 µl) was loaded onto a C18 precolumn (300 μm inner diameter × 5 mm; 5 µm particule size; 100 Å pore size; Dionex) at 20 μl/min in 5% acetonitrile, 0.05% trifluoroacetic acid. After 5 min of desalting, the precolumn was switched online with the analytical C18 column (75 μm inner diameter × 50 cm; 3 µm particule size; 120 Å pore size in-house; packed with Reprosil C18) and was equilibrated in 95% solvent A (5% acetonitrile, 0.2% formic acid) and 5% solvent B (80% acetonitrile, 0.2% formic acid). Peptides were eluted using a 5–50% gradient of solvent B over 110 min at a flow rate of 300 nl/min. The mass spectrometer was operated in a data-dependent acquisition mode with Xcalibur software. Survey MS scans were acquired in the Orbitrap on the 300–2000 m/z range with the resolution set at 60,000. The 20 most intense ions per survey scan were selected for CID fragmentation and the resulting fragments were analyzed in the linear ion trap (LTQ). A dynamic exclusion of 60 s was used to prevent repetitive selection of the same peptide. Each sample was injected once for MS analysis.

Protein identification and quantification from raw nanoLC-MS/MS data

Raw nanoLC-MS/MS files were processed with the MaxQuant software (version 1.5.2.8) for database search with the Andromeda search engine and for quantitative analysis39. Data were searched against “Mus musculus” entries in the Swiss-Prot protein database (UniProtKB/Swiss-Prot protein knowledgebase release 2013_06; 16,641 sequence entries of Mus musculus). Carbamidomethylation of cysteine was set as a fixed modification whereas oxidation of methionine and protein N-terminal acetylation were set as variable modifications. Specificity of trypsin digestion was set for cleavage after K or R and two missed trypsin cleavage sites were allowed. The precursor mass tolerance was set to 20 ppm for the first search and 4.5 ppm for the main Andromeda database search. The mass tolerance in MS/MS mode was set to 0.8 Da. Minimum peptide length was set to 7 amino acids and minimum number of unique peptides was set to 1. Andromeda results were validated by the target-decoy approach using a reverse database at both a peptide and protein FDR of 1%. For label-free relative quantification of the samples, the “match between runs” option of MaxQuant was enabled with a time window of 3 min to cross-assign the MS features detected in the different runs.

Data processing and statistical analysis

Protein entries identified as potential contaminants from the ‘proteinGroups.txt’ files generated by MaxQuant were eliminated from the analysis, as were proteins identified by fewer than two peptides. Protein relative quantification was performed by comparisons of different groups of eight samples each (8 biological replicates per group: WT, DKD, DKD + R, WT + R) (Table S1). Protein intensities were normalized across all conditions by the median intensity. For each comparison, only proteins which were quantified in at least 4 biological replicates (4 intensities values retrieved by MaxQuant) in at least one of the groups were considered for further processing and statistical analysis (Filter 1, columns AR to AU, Table S1). Remaining missing values were then replaced by a constant noise value determined independently for each analytical run as the 1% percentile of the total protein population. The mean proportion of missing values over the whole analytic run was 2.1% (line 2,435, column S, Table S1). Proteins with a p value of less than 0.05 (T-test) were considered as significantly varying between two groups.

Proteomic data availability

The mass spectrometry proteomics raw data have been deposited to the ProteomeXchange Consortium via the PRIDE40 partner repository with the dataset identifier PXD018728.

Pathway enrichment analysis

Pathway enrichment analysis of up- and down-regulated proteins was using the Gene Set Enrichement Analysis (GSEA) software package from the Molecular Signatures Database (MSigDB) (https://www.gsea-msigdb.org)10,41,42,43 using “Hallmarks gene sets” and “Canonical Pathways” as Compute Overlaps. MSigDB is a joint project of UC San Diego and Broad Institute.

Connectivity Map analysis

The initial version of CMap (CMap1: https://portals.broadinstitute.org/cmap) consists of 6,100 differential expression profiles obtained after treatment of 3 cultured human cells (MCF7, PC3, and HL60) with varying concentrations of 1,309 compounds11. More recently, a new CMap version was released44 (CMap2: https://clue.io/) encompassing 8,549 differential expression profiles obtained after treatment of 9 cultured human cells (VCAP, A375, A549, HAE1, HCC515, HEPG2, HT29, MCF7, PC3) with varying concentrations of 2,428 compounds. For our experiments, each mouse protein ID was first converted to its human ortholog and then converted into human gene ID. Up- and down-gene IDs were then queried to CMap1 and CMap2 to retrieve compounds with best negative enrichment as recently recommended19.

DMAPT synthesis

Dimethylaminoparthenolide monofumarate [(13-(N,N-dimethyl)-amino-4a,5b-epoxy-4,10-dimethyl-6a-hydroxy-12-oic acid-c-lactonegermacra-1(10)-ene monofumarate)] was synthesized by reaction of parthenolide (Sigma-Aldrich) with dimethylamine (Sigma-Aldrich) and isolated as the fumarate salt as previously described21. Analytical data (1H and 13C NMR, mass spectrometry and melting point) are consistent to those previously reported16. DMAPT fumarate purity was checked by elemental analysis and was evaluated > 98% (Supplementary Methods and Figure S1).

Statistics

Comparison between 2 groups of values was implemented using a two-tailed unpaired Welch’s t-tests. Comparison between more than 2 groups, was implemented using an ordinary one-way ANOVA followed by Homl–Sidak’s multiple comparisons test was used. P < 0.05 was considered statistically significant. For the proteomic data the P values were adjusted for the false discovery rate (Benjamini–Hochberg).

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