DOI: 10.5281/zenodo.18593873
title: Processed snRNA-seq Seurat object for kidneys of the diabetic kidney disease GIPRdn-tg mouse model treated ± FC-insulin

description: This dataset contains a processed snRNA sequencing dataset stored as a Seurat object (.rds), with normalized gene expression data, cell-level metadata, dimensionality reductions (PCA and UMAP), and cell-type annotations.

Organism: Mus Musculus, CD1 background

Tissue: KidneyMice: The data were generated using the GIPR dominant negative transgenic mouse model that recapitulates key features of human diabetic kidney disease (DKD). The study assessed transcriptional responses during diabetic conditions and following glycemic normalization using a novel ultra long acting Fc conjugated insulin analog. At 7 weeks of age, wildtype mice received vehicle in parallel to GIPRdn-tg mice (reference see below), which were split into insulin and vehicle groups. Insulin-treated mice received an initial dose of 40 nmol/kg ultra-long-acting insulin (compound 12), adjusted weekly (35–20 nmol/kg) until a maintenance dose (20–25 nmol/kg) was reached. From treatment weeks 5–16, insulin was administered weekly, with glucose monitored daily initially, then twice weekly. (Reference: Herbach, N., Schairer, I., Blutke, A., Kautz, S., Siebert, A., Göke, B., Wolf, E., and Wanke, R. (2009). Diabetic kidney lesions of GIPRdn transgenic mice: podocyte hypertrophy and thickening of the GBM precede glomerular hypertrophy and glomerulosclerosis. Am J Physiol Renal Physiol 296, F819-829. 10.1152/ajprenal.90665.2008.

Sample Collection and Nuclei Isolation: At 23 weeks of age, kidneys were perfusion fixed, and a 1 mm transverse mid section containing cortex, outer/inner medulla, and papilla was collected. After trimming ~1 mm cortex from both sides, tissue sections were snap frozen for nuclei extraction. For each group (4 WT, 4 GIPRdn tg Vehicle, 4 GIPRdn tg Insulin), frozen kidney slices were homogenized in ice cold nuclei isolation buffer (0.25 M sucrose, 25 mM KCl, 5 mM MgCl₂, 20 mM Tris pH 8.0, 0.4% IGEPAL-630, 1 mM DTT, 0.15 mM spermine, 0.5 mM spermidine, phosphatase/protease inhibitors, and RNase inhibitors). Tissue was mechanically dissociated using loose and tight Dounce pestles, filtered (100 μm → 20 μm), and centrifuged at 1000 × g, 10 min, 4°C. The nuclei pellet was resuspended in DAPI containing staining buffer and inspected for integrity using fluorescence microscopy.

Fluorescence Activated Nuclei Sorting (FANS): Nuclei were sorted on a BD FACSAria III using a 70 μm nozzle. Single nuclei were gated using:•    side scatter height vs. area•    DAPI intensity for doublet exclusionSorted nuclei were collected in nuclei buffer (<1% BSA, 0.2 U/µl RNase inhibitor) and processed immediately on ice.Library Preparation and Sequencing

GEM generation and library construction were performed using the Chromium Next GEM Automated Single Cell 3′ Reagent Kit v3.1 (Dual Index, 10x Genomics). Libraries were sequenced on an Illumina NovaSeq X+ platform with 150 bp paired end Read 2.

Preprocessing and Quality Control: Reads were aligned to the mouse reference genome GRCm38 (Ensembl v102) using CellRanger v7.2.0 to obtain gene barcode matrices. Downstream processing used Seurat v4:

Quality filtering thresholds:•    <600 detected genes per nucleus (removed)•    7000 detected genes per nucleus (removed)•    30,000 UMIs (removed)•    10% mitochondrial reads (removed)

Doublet removal: DoubletFinder v2.0

Normalization: SCTransform

Integration:•    top 3000 highly variable genes•    Reciprocal PCA (RPCA)•    IntegrateData workflow

Dimensionality reduction & clustering:•    first 16 PCs•    Louvain clustering•    UMAP for visualization

Nuclei: 102.861 high-quality nuclei